Gibco Cell Culture Basics Handbook Americas FLR

Cell Culture Basics Handbook
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Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Purpose of the Handbook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Introduction to Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

What is cell culture? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Finite vs. continuous cell line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Culture conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Morphology of cells in culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Applications of cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2. Cell Culture Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Biosafety levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
SDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Safety equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Personal protective equipment (PPE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Safe laboratory practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Cell Culture Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Basic equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Expanded equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Additional supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Cell Culture Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Aseptic work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Cell culture hood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Cell culture hood layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Incubator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Cryogenic storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Cell counter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Aseptic Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Sterile work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Good personal hygiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Sterile reagents and media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Sterile handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Cell Culture Basics | iii

For educational purposes only.
Contents
Aseptic Technique Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Biological Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Molds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Mycoplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Cross-contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Using antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3. Cell Culture Basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Selecting the appropriate cell line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Acquiring cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Culture Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Adherent vs. suspension culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
CO2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Cell Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Variations in mammalian cell morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Morphology of 293 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Insect Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Morphology of Sf21 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Morphology of Sf9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
4. Cell Culture Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Guidelines for Maintaining Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

What is subculture? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
When to subculture? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Media recommendations for common cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
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Contents
Dissociating adherent cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

TrypLE™ dissociation enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Subculturing Adherent Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Materials needed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Protocol for passaging adherent cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Notes on subculturing adherent insect cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Subculturing Suspension Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Passaging suspension cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Suspension culture vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Protocol for passaging suspension cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Notes on subculturing suspension insect cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Freezing Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Guidelines for cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Freezing medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Cryopreserving cultured cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Thawing Frozen Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Guidelines for thawing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Thawing frozen cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
5. Transfection Basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Introduction to Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
What is transfection? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Types of Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Transient transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Stable transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Choosing a transfection strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Gene Delivery Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Cationic lipid-mediated delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
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Contents
Calcium phosphate co‑precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

DEAE-Dextran-mediated delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Delivery by other cationic polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Viral delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Other physical delivery methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Cationic Lipid-Mediated Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Cationic lipid transfection reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Virus-Mediated Gene Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Key properties of viral vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Common viral vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Neon® Transfection System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Selection of Stable Transfectants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Selection antibiotics for eukaryotic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Reporter Gene Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Transfection assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Gene regulation assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Common reporter genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
RNAi and Non-coding RNA Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Glossary of common RNAi terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
How RNAi works . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
siRNA analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
miRNA analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Choosing an RNAi approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
6. Transfection Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Factors Influencing Transfection Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Cell type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Cell health and viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Confluency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
vi | Cell Culture Basics

Contents
Type of molecule transfected . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Transfection method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Selecting a Transfection Method (non-viral) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Continuous cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Primary cells and finite cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Selecting a Viral DNA Delivery System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Expression in mammalian cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Expression in insect cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Guidelines for Plasmid DNA Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Vector considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Quality of plasmid DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Gene product and promoter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Optimization of Plasmid DNA Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Considerations for calcium phosphate co‑precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Considerations for cationic lipid-mediated delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Considerations for electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Selection of Stable Transfectants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Before starting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Kill curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Selection workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Selecting a RNAi Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

siRNA vs. vector approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Non-vector siRNA technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
siRNA transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Vector-mediated RNAi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Guidelines for RNA Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

RNAi workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Handling RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Transfection efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Positive controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Negative controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Co-transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
siRNA quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
vii
Contents
siRNA quantity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Volume of transfection reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Cell density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Exposure to transfection agent/siRNA complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Presence of serum during transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Tips for a successful siRNA experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Optimization of siRNA Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

Factors affecting siRNA transfection efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Cell Culture and Transfection Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Media for mammalian cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Media for insect cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Serum products for cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Laboratory reagents for cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Antibiotics and antimycotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Growth factors and purified proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Accessory products for cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Transfection reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Neon® Transfection System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
RNA interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Additional Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

Mammalian and insect cell cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Cell and tissue analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Transfection selection tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Safety data sheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Certificate of analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
viii | Cell Culture Basics

1. Introduction
Purpose of the Handbook

Cell Culture Basics Companion Handbook is a supplement to the Cell Culture Basics
instructional videos available online at www.lifetechnologies.com/cellculturebasics.
The handbook and videos are intended as an introduction to cell culture basics. The
first four chapters of the handbook focus on cell culture, covering topics such as getting
familiar with the requirements of a laboratory dedicated to cell culture experiments,
laboratory safety, aseptic technique, and microbial contamination of cell cultures,
as well as providing basic methods for passaging, freezing, and thawing cultured
cells. The subsequent two chapters of the handbook focus on various transfection
technologies and provide general guidelines for the selection of the appropriate
transfection method, the transfection of cells with plasmid DNA, oligonucleotides, and
RNA, as well as culture preparation for in vitro and in vivo transfection and selection of
the transfected cells.
The information and guidelines presented in the handbook and the instructional
videos focus on cell lines (finite or continuous) and omit experiments and techniques
concerning primary cultures and stem cells, such as isolating and disaggregating
tissues, reprogramming cells into pluripotent stem cells, or differentiating stem cells
into various lineages.
Note that while the basics of cell culture experiments share certain similarities, cell
culture conditions vary widely for each cell type. Deviating from the culture conditions
required for a particular cell type can result in different phenotypes being expressed;
we therefore recommend that you familiarize yourself with your cell line of interest,
and closely follow the instructions provided with each product you are using in your
experiments.
Cell Culture Basics | 1

Part 1. Introduction
Introduction to Cell Culture

What is cell culture? Cell culture refers to the removal of cells from an animal or plant and their subsequent
growth in a favorible artifical environment. The cells may be removed from the tissue
directly and disaggregated by enzymatic or mechanical means before cultivation, or
they may be derived from a cell line or cell strain that has already been established.
Primary culture
Primary culture refers to the stage of the culture after the cells are isolated from
the tissue and proliferated under the appropriate conditions until they occupy all
of the available substrate (i.e., reach confluence). At this stage, the cells have to be
subcultured (i.e., passaged) by transferring them to a new vessel with fresh growth
medium to provide more room for continued growth.
Cell line
After the first subculture, the primary culture becomes known as a cell line. Cell lines
derived from primary cultures have a limited life span (i.e., they are finite; see below),
and as they are passaged, cells with the highest growth capacity predominate, resulting
in a degree of genotypic and phenotypic uniformity in the population.
Cell strain
If a subpopulation of a cell line is positively selected from the culture by cloning or
some other method, this cell line becomes a cell strain. A cell strain often acquires
additional genetic changes subsequent to the initiation of the parent line.
Finite vs. continuous

cell line Normal cells usually divide only a limited number of times before losing their ability
to proliferate, which is a genetically determined event known as senescence; these cell
lines are known as finite. However, some cell lines become immortal through a process
called transformation, which can occur spontaneously or can be chemically or virally
induced. When a finite cell line undergoes transformation and acquires the ability to
divide indefinitely, it becomes a continuous cell line.
Culture conditions Culture conditions vary widely for each cell type, but the artifical environment in which
the cells are cultured invariably consists of a suitable vessel containing a substrate or
medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins,
minerals), growth factors, hormones, and gases (O2, CO2), and regulates the physico-
chemical milieu (pH, osmotic pressure, temperature). Most cells are anchorage-
dependent and must be cultured while attached to a solid or semi-solid substrate
(adherent or monolayer culture), while others can be grown floating in the culture
medium (suspension culture).
Cryopreservation If a surplus of cells are available from subculturing, they should be treated with the
appropriate protective agent (e.g., DMSO or glycerol) and stored at temperatures below
–130°C (cryopreservation) until they are needed. For more information on subculturing
and cryopreserving cells, refer to the Guidelines for Maintaining Cultured Cells,
page 26.
2 | Cell Culture Basics

Part 1. Introduction
Morphology of cells in
culture Cells in culture can be divided in to three basic categories based on their shape and
appearance (i.e., morphology).
• Fibroblastic (or fibroblast-like) cells are bipolar or multipolar, have elongated

shapes, and grow attched to a substrate.
• Epithelial-like cells are polygonal in shape with more regular dimensions, and grow
attached to a substrate in discrete patches.
• Lymphoblast-like cells are spherical in shape and usually grown in suspension

without attaching to a surface.
Applications of
cell culture Cell culture is one of the major tools used in cellular and molecular biology, providing
excellent model systems for studying the normal physiology and biochemistry of cells
(e.g., metabolic studies, aging), the effects of drugs and toxic compounds on the cells,
and mutagenesis and carcinogenesis. It is also used in drug screening and development,
and large scale manufacturing of biological compounds (e.g., vaccines, therapeutic
proteins). The major advantage of using cell culture for any of the these applications is
the consistency and reproducibility of results that can be obtained from using a batch of
clonal cells.
3
2. Cell Culture Laboratory
Safety
In addition to the safety risks common to most everyday work places, such as electrical
and fire hazards, a cell culture laboratory has a number of specific hazards associated
with handling and manipulating human or animal cells and tissues, as well as toxic,
corrosive, or mutagenic solvents and reagents. The most common of these hazards are
accidental inoculations with syringe needles or other contaminated sharps, spills and
splashes onto skin and mucous membranes, ingestion through mouth pipetting, animal
bites and scratches, and inhalation exposures to infectious aerosols.
The fundamental objective of any biosafety program is to reduce or eliminate exposure

of laboratory workers and the outside environment to potentially harmful biological
agents. The most important element of safety in a cell culture laboratory is the strict
adherence to standard microbiological practices and techniques.
Biosafety levels The regulations and recommendations for biosafety in the United States are contained
in the document Biosafety in Microbiological and Biomedical Laboratories, prepared by
the Centers for Disease Control (CDC) and the National Institues of Health (NIH),
and published by the U.S. Department of Health and Human Services. The document
defines four ascending levels of containment, referred to as biosafety levels 1 through
4, and describes the microbiological practices, safety equipment, and facility safeguards
for the corresponding level of risk associated with handling a particular agent.
Biosafety Level 1 (BSL-1)

BSL-1 is the basic level of protection common to most research and clinical laboratories,
and is appropriate for agents that are not known to cause disease in normal, healthy
humans.

BSL-2 is appropriate for moderate-risk agents known to cause human disease of varying
severity by ingestion or through percutaneous or mucous membrane exposure. Most
cell culture labs should be at least BSL-2, but the exact requirements depend upon the
cell line used and the type of work conducted.

BSL-3 is appropriate for indigenous or exotic agents with a known potential for aerosol
transmission, and for agents that may cause serious and potentially lethal infections.

BSL-4 is appropriate for exotic agents that pose a high individual risk of life-threatening
disease by infectious aerosols and for which no treatment is available. These agents are
restricted to high containment laboratories.
For more information about the biosafety level guidelines, refer to Biosafety in
Microbiological and Biomedical Laboratories, 5th Edition, which is available for downloading
at www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm.

Part 2. Cell Culture Laboratory
SDS Safety Data Sheet (SDS) is a form containing information regarding the properties of
a particular substance, including physical data such as melting point, boiling point,
and flash point, as well as information on its toxicity, reactivity, health effects, storage,
disposal, recommended protective equipment, and handling spills.
SDSs for all Life Technologies products are available at www.lifetechnologies.com/sds.
Safety equipment Safety equipment in a cell culture laboratory includes primary barriers such as
biosafety cabinets, enclosed containers, and other engineering controls designed to
remove or minimize exposure to hazardous materials, as well as personal protective
equipment (PPE) that is often used in conjuction with the primary barriers. The
biosafety cabinet (i.e., cell culture hood) is the most important equipment to provide
containment of infectious splashes or aerosols generated by many microbiological
procedures. For more information, see Cell Culture Hood, page 7.
Personal protective
equipment (PPE) Personal protective equipment (PPE) form an immediate barrier between the personnel
and the hazardous agent, and they include items for personal protection such as gloves,
laboratory coats and gowns, shoe covers, boots, respirators, face shields, safety glasses,
or goggles. They are often used in combination with biosafety cabinets and other
devices that contain the agents, animals, or materials being handled. We recommend
that you consult your institution’s guidelines for the appropriate use of PPE in your
laboratory.
Safe laboratory practices The following recommendations are simply guidelines for safe laboratory practices, and
they should not be interpreted as a complete code of practice. Consult your institution’s
safety committee and follow local rules and regulations pertaining to laboratory safety.
For more information on standard microbiological practices and for specific biosafety
level guidelines, refer to Biosafety in Microbiological and Biomedical Laboratories, 5th Edition
at www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm.
• Always wear appropriate personal protective equipment. Change gloves when

contaminated, and dispose of used gloves with other contaminated laboratory waste.
• Wash your hands after working with potentially hazardous materials and before
leaving the laboratory.
• Do not eat, drink, smoke, handle contact lenses, apply cosmetics, or store food for
human consumption in the laboratory.
• Follow the institutional policies regarding safe handling of sharps (i.e., needles,
scalpels, pipettes, and broken glassware).
• Take care to minimize the creation of aerosols and/or splashes.
• Decontaminate all work surfaces before and after your experiments, and
immediately after any spill or splash of potentially infectious material with an
appropriate disinfectant. Clean laboratory equipment routinely, even if it is not
contaminated.
• Decontaminate all cultures, stocks, and other potentially infectious materials before
disposal.
• Report any incidents that may result in exposure to infectious materials to
appropriate personnel (e.g., laboratory supervisor, safety officer).
5
Cell Culture Equipment

The specific requirements of a cell culture laboratory depend mainly on the type of
research conducted; for example, the needs of mammalian cell culture laboratory
specilizing in cancer research is quite different from that of an insect cell culture
laboratory that focuses on protein expression. However, all cell culture laboratories
have the common requirement of being free from pathogenic microorganisms (i.e.,
asepsis), and share some of the same basic equipment that is essential for culturing cells.
This section lists the equipment and supplies common to most cell culture laboratories,
as well as beneficial equipment that allows the work to be performed more efficiently
or accurately, or permits wider range of assays and analyses. Note that this list is not
all inclusive; the requirements for any cell culture laboratory depend the type of work
conducted.
Basic equipment • Cell culture hood (i.e., laminar-flow hood or biosafety cabinet)
• Incubator (humid CO2 incubator recommended)
• Water bath
• Centrifuge
• Refrigerator and freezer (–20°C)
• Cell counter (e.g., Countess® II Automated Cell Counter or hemocytometer)
• Inverted microscope
• Liquid nitrogen (N2) freezer or cryostorage container
• Sterilizer (i.e., autoclave)
Expanded equipment • Aspiration pump (peristaltic or vacuum)

• pH meter
• Roller racks (for scaling up monolayer cultures)
• Confocal microscope
• Flow cytometer
• EG bioreactors
• Cell cubes
Additional supplies • Cell culture vessels (e.g., flasks, Petri dishes, roller bottles, multiwell plates)
• Pipettes and pipettors
• Syringes and needles
• Waste containers
• Media, sera, and reagents
• Cells

Cell Culture Laboratory

Aseptic work area The major requirement of a cell culture laboratory is the need to maintain an aseptic
work area that is restricted to cell culture work. Although a separate tissue culture room
is preferred, a designated cell culture area within a larger laboratory can still be used
fort sterile handling, incubation, and storage of cell cultures, reagents, and media. The
simplest and most economical way to provide aseptic conditions is to use a cell culture
hood (i.e., biosafety cabinet).
Cell culture hood The cell culture hood provides an aseptic work area while allowing the containment of
infectious splashes or aerosols generated by many microbiological procedures. Three
kinds of cell culture hoods, designated as Class I, II and III, have been developed to
meet varying research and clinical needs.
Classes of cell culture hoods

Class I cell culture hoods offer significant levels of protection to laboratory personnel
and to the environment when used with good microbiological techniques, but they do
not provide cultures protection from contamination. They are similar in design and air
flow characteristics to chemical fume hoods.
Class II cell culture hoods are designed for work involving BSL-1, 2, and 3 materials,
and they also provide an aseptic environment necessary for cell culture experiments. A
Class II biosafety cabinet should be used for handling potentially hazardous materials
(e.g., primate-derived cultures, virally infected cultures, radioisotopes, carcinogenic or
toxic reagents).
Class III biosafety cabinets are gas-tight, and they provide the highest attainable level
of protection to personnel and the environment. A Class III biosafety cabinet is required
for work involving known human pathogens and other BSL-4 materials.
Air-flow characteristics of cell culture hoods

Cell culture hoods protect the working enviroment from dust and other airborn
contaminants by maintaining a constant, unidirectional flow of HEPA-filtered air over
the work area. The flow can be horizontal, blowing parallel to the work surface, or it
can be vertical, blowing from the top of the cabinet onto the work surface.
Depending on its design, a horizontal flow hood provides protection to the culture (if
the air flowing towards the user) or to the user (if the air is drawn in through the front
of the cabinet by negative air pressure inside). Vertical flow hoods, on the other hand,
provide significant protection to the user and the cell culture.
Clean benches
Horizontal laminar flow or vertical laminar flow “clean benches” are not biosafety
cabinets; these pieces of equipment discharge HEPA-filtered air from the back of the
cabinet across the work surface toward the user, and they may expose the user to
potentially hazardous materials. These devices only provide product protection. Clean
benches can be used for certain clean activities, such as the dust-free assembly of sterile
equipment or electronic devices, and they should never be used when handling cell
culture materials or drug formulations, or when manipulating potentially infectious
materials.
For more information on the selection, installation, and use of biosafety cabinets, refer
to to Biosafety in Microbiological and Biomedical Laboratories, 5th Edition, which is available
for downloading at www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm.
7
Cell culture hood layout A cell culture hood should be large enough to be used by one person at a time, be easily
cleanable inside and outside, have adequate lighting, and be comfortable to use without
requiring awkward positions. Keep the work space in the cell culture hood clean and
uncluttered, and keep everything in direct line of sight. Disinfect each item placed in the
cell culture hood by spraying them with 70% ethanol and wiping clean.
The arrangement of items within the cell culture hood usually adheres to the following
right-handed convention, which can be modified to include additional items used in
specific applications.
• A wide, clear work space in the center with your cell culture vessels
• Pipettor in the front right and glass pipettes in the left, where they can be reached
easily
• Reagents and media in the rear right to allow easy pipetting
• Small container in the rear middle to hold liquid waste
Wrapped
Disposable
Pipettes
Glass Pipettes Waste Liquid

1X
(if using)
DPBS DMEM
Pipettor
Cell Culture Flasks

Reagents and Media
Work Surface
Waste Container
Figure 2.1 The basic layout of a cell culture hood for right-handed workers. Left-handed workers may
switch the positions of the items laid out on the work surface.

Incubator The purpose of the incubator is to provide the appropriate environment for cell growth.
The incubator should be large enough, have forced-air circulation, and should have
temperature control to within ±0.2°C. Stainless steel incubators allow easy cleaning
and provide corrosion protection, especially if humid air is required for incubation.
Although the requirement for aseptic conditions in a cell culture incubator is not as
stringent as that in a cell culture hood, frequent cleaning of the incubator is essential to
avoid contamination of cell cultures.
Types of incubators
There are two basic types of incubators, dry incubators and humid CO2 incubators. Dry
incubators are more economical, but require the cell cultures to be incubated in sealed
flasks to prevent evaporation. Placing a water dish in a dry incubator can provide
some humidity, but they do not allow precise control of atmospheric conditions in
the incubator. Humid CO2 incubators are more expensive, but allow superior control
of culture conditions. They can be used to incubate cells cultured in Petri dishes or
multiwell plates, which require a controlled atmosphere of high humidity and increased
CO2 tension.
Storage A cell culture laboratory should have storage areas for liquids such as media and
reagents, for chemicals such as drugs and antibiotics, for consumables such as
disposable pipettes, culture vessels, and gloves, for glassware such as media bottles and
glass pipettes, for specialized equipment, and for tissues and cells.
Glassware, plastics, and specilized equipment can be stored at ambient temperature

on shelves and in drawers; however, it is important to store all media, reagents, and
chemicals according to the instructions on the label.
Some media, reagents, and chemicals are sensitive to light; while their normal
laboratory use under lighted conditions is tolerated, they should be stored in the dark
or wrapped in aluminum foil when not in use.
Refrigerators
For small cell culture laboratories, a domestic refrigerator (preferably one without a
autodefrost freezer) is an adequate and inexpensive piece of equipment for storing
reagents and media at 2–8°C. For larger laboratories, a cold room restricted to cell
culture is more appropriate. Make sure that the refrigerator or the cold room is cleaned
regularly to avoid contamination.
Freezers
Most cell culture reagents can be stored at –5°C to –20°C; therefore an ultradeep
freezer (i.e., a –80°C freezer) is optional for storing most reagents. A domestic freezer
is a cheaper alternative to a laboratory freezer. While most reagents can withstand
temperature oscillations in an autodefrost (i.e., self-thawing) freezer, some reagents such
as antibiotics and enzymes should be stored in a freezer that does not auto-defrost.
9
Cryogenic storage Cell lines in continuous culture are likely to suffer from genetic instability as their
passage number increases; therefore, it is essential to prepare working stocks of the cells
and preserve them in cryogenic storage (for more information, see Freezing Cells,
page 37). Do not store cells in –20°C or –80°C freezers, because their viability quicky
decreases when they are stored at these temperatures.
There are two main types of liquid-nitrogen storage systems, vapor phase and liquid
phase, which come as wide-necked or narrow-necked storage containers. Vapor phase
systems minimize the risk of explosion with cryostorage tubes, and are required for
storing biohazardous materials, while the liquid phase systems usually have longer
static holding times, and are therefore more economical.
Narrow-necked containers have a slower nitrogen evaporation rate and are more
economical, but wide-necked containers allow easier access and have a larger storage
capacity.
Cell counter A cell counter is essential for quantitative growth kinetics, and a great advantage when
more than two or three cell lines are cultured in the laboratory.
The Countess® II Automated Cell Counter is a benchtop instrument designed to

measure cell count and viability (live, dead, and total cells) accurately and precisely
in less than a minute per sample, using the standard Trypan Blue uptake technique.
Using the same amount of sample that you currently use with the hemocytometer, the
Countess® II Automated Cell Counter takes less than a minute per sample for a typical
cell count and is compatible with a wide variety of eukaryotic cells.

Aseptic Technique
Introduction Successful cell culture depends heavily on keeping the cells free from contamination by
microorganisms such as bacterial, fungi, and viruses. Nonsterile supplies, media, and
reagents, airborne particles laden with microorganisms, unclean incubators, and dirty
work surfaces are all sources of biological contamination.
Aseptic technique, designed to provide a barrier between the microrganisms in the

environment and the sterile cell culture, depends upon a set of procedures to reduce
the probability of contamination from these sources. The elements of aseptic technique
are a sterile work area, good personal hygiene, sterile reagents and media, and sterile
handling.
Sterile work area The simplest and most economical way to reduce contamination from airborne particles
and aerosols (e.g., dust, spores, shed skin, sneezing) is to use a cell culture hood.
• The cell culture hood should be properly set up, and be located in an area that is
restricted to cell culture that is free from drafts from doors, windows, and other
equipment, and with no through traffic.
• The work surface should be uncluttered and contain only items required for a
particular procedure; it should not be used as a storage area.
• Before and after use, the work surface should be disinfected thoroughly, and the
surrounding areas and equipment should be cleaned routinely.
• For routine cleaning, wipe the work surface with 70% ethanol before and during
work, especially after any spillage.
• Using a Bunsen burner for flaming is not necessary nor recommended in a cell
culture hood.
• Leave the cell culture hood running at all times, turning them off only when they
will not be used for extended periods of time.
Good personal hygiene Wash your hands before and after working with cell cultures. In addition to protecting
you from hazardous materials, wearing personal protective equipment also reduces the
probability of contamination from shed skin as well as dirt and dust from your clothes.
11
Sterile reagents and

media Commercial reagents and media undergo strict quality control to ensure their sterility,
but they can become contaminated while handling. Follow the guidelines below for
sterile handling to avoid contaminating them. Always sterilize any reagents, media, or
solutions prepared in the laboratory using the appropriate sterilization procedure (e.g.,
autoclave, sterile filter).
Sterile handling • Always wipe your hands and your work area with 70% ethanol.
• Wipe the outside of the containers, flasks, plates, and dishes with 70% ethanol before
placing them in the cell culture hood.
• Avoid pouring media and reagents directly from bottles or flasks.
• Use sterile glass or disposable plastic pipettes and a pipettor to work with liquids,
and use each pipette only once to avoid cross contamination. Do not unwrap sterile
pipettes until they are to be used. Keep your pipettes at your work area.
• Always cap the bottles and flasks after use and seal multi-well plates with tape or
place them in resealable bags to prevent microorganisms and airborn contaminants
from gaining entry.
• Never uncover a sterile flask, bottle, petri dish, etc. until the instant you are ready to
use it and never leave it open to the environment. Return the cover as soon as you
are finished.
• If you remove a cap or cover, and have to put it down on the work surface, place the
cap with opening facing down.
• Use only sterile glassware and other equipment.
• Be careful not to talk, sing, or whistle when you are performing sterile procedures.
• Perform your experiments as rapidly as possible to minimize contamination

Aseptic Technique Checklist

The following checklist provides a concise list of suggestions and procedures to guide
you to achieve a solid aseptic technique. For an in-depth review of aseptic technique,
refer to Culture of Animal Cells: A Manual of Basic Technique (Freshney, 2000).
Work Area
Is the cell culture hood properly set up?
Is the cell culture hood in an area free from drafts and through traffic?
Is the work surface uncluttered, and does it contain only items required for your
experiment?
Did you wipe the work surface with 70% ethanol before work?
Are you routinely cleaning and sterilizing your incubators, refrigerators, freezers,
and other laboratory equipment?
Personal Hygiene
Did you wash your hands?
Are you wearing personal protective equipment?
If you have long hair, is it tied in the back?
Are you using a pipettor to work with liquids?
Reagents and Media
Have you sterilized any reagents, media, and solutions you have prepared in the
laboratory using the appropriate procedure?
Did you wipe the outside of the bottles, flasks, and plates with 70% ethanol before
placing them on your work surface?
Are all your bottles, flasks, and other containers capped when not in use?
Are all your plates stored in sterile re-sealeable bags?
Does any of your reagents look cloudy? Contaminated? Do they contain floating
paticles? Have foul smell? Unusual color? If yes, did you decontaminate and discard
them?
Handling
Are you working slowly and deliberately, mindful of aseptic technique?
Did you wipe the surfaces of all the items, including pipettor, bottles, flasks with
70% ethanol before placing them in the cell culture hood?
Are you placing the caps or covers face down on the work area?
Are you using sterile glass pipettes or sterile disposable plastic pipettes to
manipulate all liquids?
Are you using a sterile pipette only once to abvoid cross contamination?
Are you careful not to touch the pipette tip to anything nonsterile?
Did you mop up any spillage immediately, and wiped the area with 70% ethanol?
13
Biological Contamination
Introduction Contamination of cell cultures is easily the most common problem encountered in
cell culture laboratories, sometimes with very serious consequences. Cell culture
contaminants can be divided into two main categories, chemical contaminants such
as impurities in media, sera, and water, endotoxins, plasticizers, and detergents, and
biological contaminants such as bacteria, molds, yeasts, viruses, mycoplasma, as
well as cross contamination by other cell lines. While it is impossible to eliminate
contamination entirely, it is possible to reduce its frequency and seriousness by gaining
a thorough understanding of their sources and by following good aseptic technique.
This section provides an overview of major types of biological contamination.
Bacteria Bacteria are a large and ubiquitious group of unicellular microorganisms. They are
typically a few micrometers in diameters, and can have a variety of shapes, ranging
from spheres to rods and spirals. Because of their ubiquity, size, and fast growth rates,
bacteria, along with yeasts and molds, are the most commonly encountered biological
contaminants in cell culture. Bacterial contamination is easily detected by visual
inspection of the culture within a few days of it becoming infected; infected cultures
usually appear cloudy, sometimes with a thin film on the surface. Sudden drops in
the pH of the culture medium is also a frequently encountered. Under a low-power
microscope, the bacteria appear as tiny granules between the cells, and observation
under a high-power microscope can resolve the shapes of individual bacteria. The
simulated images below show an adherent 293 cell culture contaminated with E. coli.
A B
Figure 2.2 Simulated phase contrast images of adherent 293 cells contaminated with E. coli. The spaces
between the adherent cells show tiny, shimmering granules under low power microscopy, but the individual
bacteria are not easily distinguishable (panel A). Further magnification of the area enclosed by the black
square resolves the individual E. coli cells, which are typically rod-shaped and are about 2 μm long and
0.5 μm in diameter. Each side of the black square in panel A is 100 μm.

Yeasts Yeasts are unicellular eukaryotic microorganisms in the kingdom of Fungi, ranging in
size from a few micrometers (typically) up to 40 micrometers (rarely). Like bacterial
contamination, cultures contaminated with yeasts become turbid, especially if the
contamination is in an advanced stage. There is very little change in the pH of the
culture contaminated by yeasts until the contamination becomes heavy, at which
stage the pH usually increases. Under microscopy, yeast appear as individual ovoid
or spherical particles, that may bud off smaller particles. The simulated image below
shows adherent 293 cell culture 24 hours after plating that is infected with yeast.
Figure 2.3 Simulated phase contrast images of 293 cells in adherent culture that is contaminated with yeast.
The contaminating yeast cells appear as ovoid particles, budding off smaller particles as they replicate.
Molds Molds are eukaryotic microorganisms in the kingdom of fungi that grow as
multicellular filaments called hyphae. A connected network of these multicellular
filaments contain genetically identical nuclei, and are referred to as a colony or
mycelium. Similar to yeast contamination, the pH of the culture remains stable in the
initial stages of contamination, then rapidly increases as the culture become more
heavily infected and becomes turbid. Under microscopy, the mycelia usually appear
as thin, wisp-like filaments, and sometimes as denser clumps of spores. Spores of
many mold species can survive extremely harsh and inhospitable environments in
their dormant stage, only to become activated when they encounter suitable growth
conditions.
15
Viruses Viruses are microscopic infectious agents that take over the host cells machinery to
reproduce. Their extremely small size makes them very difficult to detect in culture, and
to remove them from reagents used in cell culture laboratories. Because most viruses
have very stringent requirements for their host, they usually do not adversely effect
cell cultures from species other than their host. However, using virally infected cell
cultures can present a serious health hazard to the laboratory personnel, especially if
human or primate cells are cultured in the laboratory. Viral infection of cell cultures can
be detected by electron microscopy, immunostaining with a panel of antibodies, ELISA
assays, or PCR with appropriate viral primers.
Mycoplasma Mycoplasma are simple bacteria that lack a cell wall, and they are considered the
smallest self-replicating organism. Because of their extremely small size (typically
less than one micrometer), mycoplasma are very difficult to detect until they achieve
extremely high densities and cause the cell culture to deteriorate; until then, there are
often no visible signs of infection. Some slow growing mycoplasma may persists in
culture without causing cell death, but they can alter the behavior and metabolism of
the host cells in the culture. Chronic mycoplasma infections might manifest themselves
with decreased rate of cell proliferation, reduced saturation density, and agglutination
in suspension cultures; however, the only assured way of detecting mycoplasma
contamination is by testing the cultures periodically using fluorescent staining (e.g.,
Hoechst 33258), ELISA, PCR, immunostaining, autoradiography, or microbiological
assays.
A B C
Figure 2.4 Photomicrographs of mycoplasma-free cultured cells (panel A) and cells infected with
mycoplasma (panels B and C). The cultures were tested using the MycoFluor™ Mycoplasma Detection Kit,
following the kit protocols. In fixed cells, the MycoFLuor™ reagent has access to the cell nuclei, which are
intesensely stained with the reagent, but the absence of fluorescent extranuclear objects indicates that
the culture is free from mycoplasma contamination (panel A). In fixed cells infected with mycoplasma, the
MycoFluor™ reagent stains both the nuclei and the mycoplasma, but the intense relative fluorescence of the
nuclei obscure the mycoplasma on or near the nuclei. However, the mycoplasma separated from the bright
nuclei are readily visible (panel B). In live cells, the MycoFluor™ reagent does not have access to the nuclei,
but readily stains the mycoplasma associated with the outside of cells (panel C). The emission spectra of
the MORFS are designed to have a homogeneous intensity that closely matches that of mycoplasma stained
according to the MycoFluor™ mycoplasma detection protocol, allowing the researchers to discriminate
between stained mycoplasma and other forms of background luminescence, including viruses, bacteria and
cellular autofluorescence. The images were obtained using 365 nm excitation and a 100/1.3 Plan Neofluar
objective lens coupled with a 450 ± 30 nm bandpass filter.

Cross-contamination While not as common as microbial contamination, extensive cross-contamination of

many cell lines with HeLa and other fast growing cell lines is a clearly-established
problem with serious consequences. Obtaining cell lines from reputable cell banks,
periodically checking the characteristics of the cell lines, and practicing good
aseptic technique are practices that will help you avoid cross-contamination. DNA
fingerprinting, karyotype analysis, and isotype analysis can confirm the presence or
absence of cross-contamination in your cell cultures.
Using antibiotics Antibiotics should never be used routinely in cell culture, because their continuous
use encourages the development of antibiotic resistant strains and allows low-level
contamination to persist, which can develop into full-scale contamination once the
antibiotic is removed from media, and may hide mycoplasma infections and other
cryptic contaminants. Further, some antibiotics might cross react with the cells and
interfere with the cellular processes under investigation.
Antibiotics should only be used as a last resort and only for short term applications,
and they should be removed from the culture as soon as possible. If they are used in
the long term, antibiotic-free cultures should be maintained in parallel as a control for
cryptic infections.
17
3. Cell Culture Basics
This section provides information on the fundamentals of cell culture, including the
selection of the appropriate cell line for your experiments, media requirements for cell
culture, adherent versus suspension culture, and morphologies of continuous cell lines
available from Life Technologies™.
Note that the following information is an introduction to the basics of cell culture, and it
is intented as a starting point in your investigations. For more in-depth information, we
recommend that you consult published literature and books, as well as the manuals and
product information sheets provided with the products you are using.
Cell Lines
Selecting the appropriate
cell line Consider the following criteria for selecting the appropriate cell line for your experiments:
• Species: Non-human and non-primate cell lines usually have fewer biosafety
restrictions, but ultimately your experiments will dictate whether to use species-
specific cultures or not.
• Functional characteristics: What is the purpose of your experiments? For example,
liver- and kidney-derived cell lines may be more suitable for toxicity testing.
• Finite or continuous: While choosing from finite cell lines may give you more
options to express the correct functions, continous cell lines are often easier to clone
and maintain.
• Normal or transformed: Transformed cell lines usually have an increased growth
rate and higher plating efficiency, are continuous, and require less serum in media,
but they have undergone a permanent change in their phenotype through a genetic
transformation.
• Growth conditions and characteristics: What are your requirements with respect
to growth rate, saturation density, cloning efficiency, and the ability to grow in
suspension? For example, to express a recombinant protein in high yields, you
might want to choose a cell line with a fast growth rate and an ability to grow in
suspension.
• Other criteria: If you are using a finite cell line, are there sufficient stocks available?
Is the cell line well characterized, or do you have the perform the validation
yourself? If you are using an abnormal cell line, do you have an equivalent normal
cell line that you can use as a control? Is the cell line stable? If not, how easy it is to
clone it and generate sufficient frozen stocks for your experiements?
Acquiring cell lines You may establish your own culture from primary cells, or you may choose to buy
established cell cultures from commercial or non-profit suppliers (i.e., cell banks).
Reputable suppliers provide high quality cell lines that are carefully tested for
their integrity and to ensure that the culture is free from contaminants. We advise
against borrowing cultures from other laboratories because they carry a high risk of
contamination. Regardless of their source, makes sure that all new cell lines are tested
for mycoplasm contamination before you begin to use them.
Life Technologies™ offers a variety of primary cultures and established cell lines,
reagents, media, sera, and growth factors for your cell culture experiments. The
Appendix section contains a list of the more commonly used cell lines available from
Life Technologies™ (see page 97). For more information on Life Technologies™ and
Gibco® products, refer to www.lifetechnologies.com.

Part 3. Cell Culture Basics
Culture Environment
One of the major advantages of cell culture is the ability to manipulate the
physiochemical (i.e., temperature, pH, osmotic pressure, O2 and CO2 tension) and
the physiological environment (i.e., hormone and nutrient concentrations) in which
the cells propagate. With the exception of temperature, the culture environment is
controlled by the growth media.
While the physiological environment of the culture is not as well defined as its
physiochemical environment, a better understanding of the components of serum, the
identification of the growth factors necessary for proliferation, and a better appreciation
of the microenvironment of cells in culture (i.e., cell-cell interactions, diffusion of gases,
interactions with the matrix) now allow the culture of certain cell lines in serum-free
media.
Adherent vs. suspension

culture There are two basic systems for growing cells in culture, as monolayers on an artificial
substrate (i.e., adherent culture) or free-floating in the culture medium (suspension
culture). The majority of the cells derived from vertebrates, with the exception of
hemopoietic cell lines and a few others, are anchorage-dependent and have to be
cultured on a suitable substrate that is specifically treated to allow cell adhesion and
spreading (i.e., tissue-culture treated). However, many cell lines can also be adapted
for suspension culture. Similarly, most of the commercially available insect cell lines
grow well in monolayer or suspension culture. Cells that are cultured in suspension
can be maintained in culture flasks that are not tissue-culture treated, but as the culture
volume to surface area is increased beyond which adequate gas exchange is hindered
(usually 0.2–0.5 mL/cm2), the medium requires agitation. This agitation is usually
achieved with a magnetic stirrer or rotating spinner flasks.
Adherent Culture Suspension Culture
Appropriate for cells adapted to suspension

Appropriate for most cell types, including
culture and a few other cell lines that are
primary cultures.
nonadhesive (e.g., hematopoietic).
Easier to passage, but requires daily cell

Requires periodic passaging, but allows
counts and viability determination to follow
easy visual inspection under inverted
growth patterns; culture can be diluted to
microscope.
stimulate growth.
Cells are dissociated enzymatically (e.g., Does not require enzymatic or mechanical
TrypLE™ Express, trypsin) or mechanically. dissocation.
Growth is limited by surface area, which Growth is limited by concentration of cells

may limit product yields. in the medium, which allows easy scale-up.
Can be maintained in culture vessels that

are not tissue-culture treated, but requires
Requires tissue-culture treated vessel.
agitation (i.e., shaking or stirring) for
adequate gas exhange.
Used for cytology, harvesting products

Used for bulk production, batch harvesting,
continuously, and many research
and many research applications.
applications.
19
Media The culture medium is the most important component of the culture environment,
because it provides the necessary nutrients, growth factors, and hormones for cell
growth, as well as regulating the pH and the osmotic pressure of the culture.
Although initial cell culture experiements were performed using natural media
obtained from tissue extracts and body fluids, the need for standardization and media
quality, as well as an increased demand led to the development of chemically defined
media. The three basic classes of media are basal media, reduced-serum media, and
serum-free media, which differ in their requirement for supplementation with serum.
Serum is vitally important as a source of growth and adhesion factors, hormones, lipids
and minerals for the culture of cells in basal media. In addition, serum also regulates
cell membrane permeability and serves as a carrier for lipids, enzymes, micronutrients
and trace elements into the cell. However, using serum in media has a number of
disadvantages including high cost, problems with standardization, specificity, and
variability, and unwanted effects such as stimulation or inhibition of growth and/or
cellular function on certain cell cultures. If the serum is not obtained from reputable
source, contamination can also pose a serious threat to successful cell culture
experiments. All Life Technologies™ and Gibco® products, including sera, are tested
for contamination and guaranteed for their quality, safety, consistency, and regulatory
compliance.
Basal media
The majority of cell lines grow well in basal media, which contain amino acids,
vitamins, inorganic salts, and a carbon source such as glucose, but these basal media
formulations must be further supplemented with serum.
Reduced-serum media
Another strategy to reduce the undesired effects of serum in cell culture experiments
is to use reduced-serum media. Reduced-serum media are basal media formulations
enriched with nutrients and animal-derived factors, which reduce the amount of serum
that is needed.
Serum-free media
Serum-free media (SFM) circumvents issues with using animal sera by replacing the
serum with appropriate nutritional and hormonal formulations. Serum-free media
formulations exist for many primary cultures and cell lines, including recombinant
protein producing lines of Chinese Hamster Ovary (CHO), various hybridoma cell
lines, the insect lines Sf9 and Sf21 (Spodoptera frugiperda), and for cell lines that act as
hosts for viral production, such as 293, VERO, MDCK, MDBK, and others. One of the
major advantages of using serum-media is the ability to make the medium selective for
specific cell types by choosing the appropriate combination of growth factors. The table
below lists the advantages and disadvantages of serum-free media.
Advantages Disadvantages
• Increased definition • Requirement for cell type-specific media

• More consistent performance formulations
• Easier purification and downstream • Need for higher degree of reagent purity
processing • Slower growth
• Precise evaluation of cellular functions
• Increased productivity
• Better control over physiological
response
• Enhanced detection of cellular mediators

Life Technologies™ offers a wide range of classical basal media, reduced-serum media,
and serum-free media, as well as sera, growth factors, supplements, antibiotics, and
reagents for your cell culture experiments. The Appendix section contains a list of the
more commonly used cell culture products available from Life Technologies™.
For more information on Life Technologies™ and Gibco® cell culture products, refer to
www.lifetechnologies.com.
pH Most normal mammalian cell lines grow well at pH 7.4, and there is very little
variability among different cell strains. However, some transformed cell lines have
been shown to grow better at slightly more acidic environments (pH 7.0–7.4), and some
normal fibroblast cell lines prefer slightly more basic environments (pH 7.4–7.7). Insect
cell lines such as Sf9 and Sf21 grow optimally at pH 6.2.
CO2 The growth medium controls the pH of the culture and buffers the cells in culture
against changes in the pH. Usually, this buffering is achieved by including an organic
(e.g., HEPES) or CO2-bicarbonate based buffer. Because the pH of the medium is
dependent on the delicate balance of dissolved carbondioxide (CO2) and bicarbonate
(HCO3–), changes in the atmospheric CO2 can alter the pH of the medium. Therefore, it
is necessary to use exogeneous CO2 when using media buffered with a CO2-bicarbonate
based buffer, especially if the cells are cultured in open dishes or transformed cell lines
are cultured at high concentrations. While most researchers usually use 5–7% CO2 in air,
4–10% CO2 is common for most cell culture experiments. However, each medium has a
recommended CO2 tension and bicarbonate concentration to achieve the correct pH and
osmolality; refer to the media manufacturer’s instructions for more information.
Temperature The optimal temperature for cell culture largely depends on the body temperature
of the host from which the cells were isolated, and to a lesser degree on the
anatomical variation in temperature (e.g., temperature of the skin may be lower than
the temperature of skeletal muscle). Overheating is a more serious problem than
underheating for cell cultures; therefore, often the temperture in the incubator is set
slightly lower than the optimal temperature.
• Most human and mammalian cell lines are maintained at 36°C to 37°C for optimal
growth.
• Insect cells are cultured at 27°C for optimal growth; they grow more slowly at lower
temperatures and at temperatures between 27°C and 30°C. Above 30°C, the viability
of insect cells decreases, and the cells do not recover even after they are returned to
27°C.
• Avian cell lines require 38.5°C for maximum growth. Although these cells can also
be maintained at 37°C, they will grow more slowly.
• Cell lines derived from cold-blooded animals (e.g., amphibians, cold-water fish)
tolerate a wide temperature range between 15°C and 26°C.
Note that cell culture conditions vary for each cell type. The consequences of deviating
from the culture conditions required for a particular cell type can range from the
expression of aberrant phenotypes to a complete failure of the cell culture. We therefore
recommend that you familiarize yourself with your cell line of interest, and closely
follow the instructions provided with each product you are using in your experiments.
21
Cell Morphology
Regularly examining the morphology of the cells in culture (i.e., their shape and
appearance) is essential for successful cell culture experiments. In addition to
confirming the healthy status of your cells, inspecting the cells by eye and a microscope
each time they are handled will allow you to detect any signs of contamination early on
and to contain it before it spreads to other cultures around the laboratory.
Signs of deterioration of cells include granularity around the nucleus, detachment of

the cells from the substrate, and cytoplasmic vacuolation. Signs of deterioriation may be
caused by a variety of reasons, including contamination of the culture, senescence of the
cell line, or the presence of toxic substances in the medium, or they may simply imply
that the culture needs a medium change. Allowing the deterioration to progress too far
will make it irreversible.
Mammalian Cells
Variations in mammalian
cell morphology Most mammalian cells in culture can be divided in to three basic categories based on
their morphology.
• Fibroblastic (or fibroblast-like) cells are bipolar or multipolar and have elongated
shapes. They grow attached to a substrate.
• Epithelial-like cells are polygonal in shape with more regular dimensions, and grow
attached to a substrate in discrete patches.
• Lymphoblast-like cells are spherical in shape and they are usually grown in
suspension without attaching to a surface.
In addition to the basic categories listed above, certain cells display morphological
characteristics specific to their specialized role in host.
• Neuronal cells exist in different shapes and sizes, but they can roughly be divided
into two basic morphological categories, type I with long axons used to move signals
over long distances and type II without axons. A typical neuron projects cellular
extensions with many branches from the cell body, which is referred to as a dendritic
tree. Neuronal cells can be unipolar or pseudounipolar with the dendrite and axon
emerging from same process, bipolar with the axon and single dendrite on opposite
ends of the soma (the central part of the cell containing the nucleus), or multipolar
with more than two dendrites.

Morphology of 293 cells The 293 cell line is a permanent line established from primary embryonic human
kidney, which was transformed with sheared human adenovirus type 5 DNA. The
adenoviral genes expressed in this cell line allow the cells to produce very high levels
of recombinant proteins. Life Technologies™ offers several variants of the 293 cell line,
including those adapted for high-density suspension culture in serum-free media. For
more information, visit our mammalian cell culture pages on our website.
The phase contrast images below show the morphology of healthy 293 cells in adherent
culture at 80% confluency (Figure 3.1) and in suspension culture (Figure 3.2). Note
that adherent mammalian cultures should be passaged when they are in the log phase,
before they reach confluence (see When to subculture, page 27).
A B
Figure 3.1 Phase contrast images of healthy 293 cells in adherent culture. The cells were plated at a seeding
density of 5 × 104 viable cells/cm2 in 293 SFM II medium and grown as a monolayer in a 37°C incubator with
a humidified atmosphere of 5% CO2 in air. The images were obtained using 10X and 20X objectives (panels
A and B, respectively) 4 days after plating.
A B
Figure 3.2 Phase contrast images of healthy 293F cells grown is suspension. The culture was started in
a shake flask at a seeding density of 2 × 105 viable cells/mL in 293 SFM II medium and grown in a 37°C
incubator with a humidified atmosphere of 5% CO2 in air. 4 days after seeding, the cells were diluted 1:3,
and the images were obtained using 10X and 20X objectives (panels A and B, respectively).
23
Insect Cells
Morphology of Sf21 cells Sf21 cells (IPLB-Sf21-AE) are ovarian cells isolated from Spodoptera frugiperda (Fall
Armyworm). They are spherical in shape with unequal sizes, and have a somewhat
granular appearance. Sf21 cells can be thawed and used directly in suspension culture
for rapid expansion of cell stocks, propagation of baculovirus stocks, and production of
recombinant proteins. Because Sf21 cells attach firmly to surfaces, they can be used as a
monolayer for transfection or plaque assay applications.
The images below show the morphology of healthy Sf21 insect cells in suspension culture
(Figure 3.3) and in adherent culture at confluency (Figure 3.4). Note that insect cells
should be subcultured when they reach confluency (see When to Subculture, 27).
A B
Figure 3.3 Phase contrast images of healthy Sf21 insect cells grown is suspension. The culture was started
in a shake flask at a seeding density of 3 × 105 viable cells/mL in Sf-900 II SFM medium and it was maintained
in a 28°C, non-humidified, ambient air-regulated incubator. The images were obtained using 10X and 20X
objectives (panels A and B, respectively) 3 days after seeding.
A B
Figure 3.4 Phase contrast images of Sf21 insect cells grown as an adherent monolayer in 293 SFM II medium.
The cells were plated at a seeding density of 5 × 104 viable cells/cm2 in a T-25 flask and grown as monolayers
objectives (panels A and B, respectively) 7 days after seeding, when the culture had reached confluency.

Morphology of Sf9 cells The Sf9 insect cell line is a clonal isolate derived from the parental Spodoptera frugiperda
cell line IPLB-Sf-21-AE, and it is a suitable host for expression of recombinant proteins
from baculovirus expression systems (e.g., Life Technologies™’ Bac-to-Bac® and
Bac‑N‑Blue™ Expression Systems). Although insect cells have been historically cultured
in stationary systems utilizing T-flasks and serum-supplemented basal medium, insect
cells are generally not anchorage dependent and can easily be maintained in suspension
culture.
The images below show the morphology of healthy Sf9 insect cells in suspension and
adherent cultures. Sf9 cells attach firmly to surfaces, and their small, regular size makes
them exceptional for the formation of monolayers and plaques.
A B
Figure 3.5 Phase contrast images of healthy Sf9 insect cells grown is suspension. The culture was started in
a shake flask at a seeding density of 3 × 105 viable cells/mL in Sf-900 II SFM medium and it was maintained
A B
Figure 3.6 Phase contrast images of healthy Sf9 insect cells grown is suspension. The culture was started in
a shake flask at a seeding density of 3 × 105 viable cells/mL in Sf-900 II SFM medium and it was maintained
25
4. Cell Culture Methods
This section provides guidelines and general procedures for routine subculturing,
thawing, and freezing of cells in culture. Note that cell culture conditions vary for each
cell type. The consequences of deviating from the culture conditions required for a
particular cell type can range from the expression of aberrant phenotypes to a complete
failure of the cell culture. We therefore recommend that you familiarize yourself with
your cell line of interest, and closely follow the instructions provided with each product
you are using in your experiments.
Guidelines for Maintaining Cultured Cells

What is subculture? Subculturing, also referred to as passaging, is the removal of the medium and transfer
of cells from a previous culture into fresh growth medium, a procedure that enables the
further propagation of the cell line or cell strain.
The growth of cells in culture proceeds from the lag phase following seeding to the log
phase, where the cells proliferate exponentially. When the cells in adherent cultures
occupy all the available substrate and have no room left for expansion, or when the cells
in suspension cultures exceed the capacity of the medium to support further growth,
cell proliferation is greatly reduced or ceases entirely (see Figure 4.1, below). To keep
the culture at an optimal density for continued cell growth and to stimulate further
proliferation, the culture has to be divided and fresh medium supplied.
106 Subculture
Cell density
105
Log phase growth

104
0 2 4 6 8 10
Days
Figure 4.1 Characteristic growth pattern of cultured cells. The semi-logarithmic plot shows the cell density
versus the time spent in culture. Cells in culture usually proliferate following a standard growth pattern. The
first phase of growth after the culture is seeded is the lag phase, which is a period of slow growth when the
cells are adapting to the culture environment and preparing for fast growth. The lag phase is followed by
the log phase (i.e., “logarithmic” phase), a period where the cells proliferate exponentially and consume the
nutrients in the growth medium. When all the growth medium is spent (i.e., one or more of the nutrients is
depleted) or when the cells occupy all of the available substrate, the cells enter the stationary phase (i.e.,
plateau phase), where the proliferation is greately reduced or ceases entirely.

Part 4. Cell Culture Methods
When to subculture? The criteria for determining the need for subculture are similar in adherent and
suspension cultures; however, there are some differences between mammalian and
insect cell lines.
Cell density
• Mammalian cells: Adherent cultures should be passaged when they are in the log
phase, before they reach confluence. Normal cells stop growing when they reach
confluence (contact inhibition), and it takes them longer to recover when reseeded.
Transformed cells can continue proliferating even after they reach confluence, but
they usually deteriorate after about two doublings. Similarly, cells in suspension
should be passaged when they are in log-phase growth before they reach confluency.
When they reach confluency, cells in suspension clump together and the medium
appears turbid when the culture flask is swirled.
• Insect cells: Insect cells should be subcultured when they are in the log phase,
before they reach confluency. While tightly adherent insect cells can be passaged at
confluency, which allows for easier detachment from the culture vessel, insect cells
that are repeatedly passaged at densities past confluency display decreased doubling
times, decreased viabilities, and a decreased ability to attach. On the other hand,
passaging insect cells in adherent culture before they reach confluency requires
more mechanical force to dislodge them from the monolayer. When repeatedly
subcultured before confluency, these cells also display decreased doubling times and
decreased viabilities, and are considered unhealthy.
Exhaustion of medium
• Mammalian cells: A drop in the pH of the growth medium usually indicates a build
up of lactic acid, which is a by-product of cellular metabolism. Lactic acid can be
toxic to the cells, and the decreased pH can be sub-optimal for cell growth. The rate
of change of pH is generally dependent on the cell concentration in that cultures at a
high cell concentration exhaust medium faster than cells lower concentrations. You
should subculture your cells if you observe a rapid drop in pH (> 0.1–0.2 pH units)
with an increase in cell concentration.
• Insect cells: Insect cells are cultured in growth media that are usually more acidic
that those used for mammalian cells. For example, TNM-FH and Grace’s medium
used for culturing Sf9 cells has a pH of 6.2. Unlike mammalian cell cultures, the pH
rises gradually as the insect cells grow, but usually does not exceed pH 6.4. However,
as with mammalian cells, the pH of the growth medium will start falling when insect
cells reach higher densities.
Subculture schedule
Passaging your cells according to a strict schedule ensures reproducible behavior and
allows you to monitor their health status. Vary the seeding density of your cultures
until you achieve consistent growth rate and yield appropriate for your cell type
from a given seeding density. Deviations from the growth patterns thus established
usually indicate that the culture is unhealthy (e.g., deterioration, contamination) or a
component of your culture system is not functioning properly (e.g., temperature is not
optimal, culture medium too old). We strongly recommend that you keep a detailed
cell culture log, listing the feeding and subculture schedules, types of media used,
the dissociation procedure followed, split ratios, morphological observations, seeding
concentrations, yields, and any anti-biotic use.
It is best to perform experiments and other non-routine procedures (e.g., changing type
of media) according to your subculture schedule. If your experimental schedule does
not fit the routine subculture schedule, make sure that you do not passage your cells
while they are still in the lag period or when they have reached confluency and ceased
growing.
27
Media recommendations
for common cell lines Many continuous mammalian cell lines can be maintained on a relatively simple
medium such as MEM supplemented with serum, and a culture grown in MEM
can probably be just as easily grown in DMEM or Medium 199. However, when
a specialized function is expressed, a more complex medium may be required.
Information for selecting the appropriate medium for a given cell type is usually
available in published literature, and may also be obtained from the source of the cells
or cell banks.
If there is no information available on the appropriate medium for your cell type,
choose the growth medium and serum empirically or test several different media for
best results. In general, a good place to start is MEM for adherent cells and RPMI-1640
for suspension cells. The conditions listed below can be used as a guide line when
setting up a new mammalian cell culture.
Insect cells are cultured in growth media that are usually more acidic that those used for
mammalian cells such as TNM-FH and Grace’s medium
Mammalian Cell Culture
Cell Line Cell Type Species Tissue Medium*
293 fibroblast human embryonic kidney MEM and 10% FBS
3T6 fibroblast mouse embryo DMEM, 10% FBS
A549 epithelial human lung carcinoma F-12K, 10% FBS
A9 fibroblast mouse connective tissue DMEM, 10% FBS
AtT-20 epithelial mouse pituitary tumor F-10, 15% horse serum, and 2.5% FBS
BALB/3T3 fibroblast mouse embryo DMEM, 10% FBS
BHK-21 fibroblast hamster kidney GMEM, 10% FBS, or MEM, 10% FBS, and NEAA
BHL-100 epitheliall human breast McCoy'5A, 10% FBS
BT fibroblast bovine turbinate cells MEM, 10% FBS, and NEAA
Caco-2 epithelial human colon adeno carcinoma MEM, 20% FBS, and NEAA
Chang epithelial human liver BME, 10% calf serum
CHO-K1 epithelial hamster ovary F-12, 10% FBS
Clone 9 epithelial rat liver F-12K, 10% FBS
Clone M-3 epithelial mouse melanoma F-10, 15% horse serum, and 2.5% FBS
COS-1, COS-3, COS-7 fibroblast monkey kidney DMEM, 10% FBS
CRFK epithelial cat kidney MEM, 10% FBS, and NEAA
CV-1 fibroblast monkey kidney MEM, 10% FBS
D-17 epithelial dog osteosarcoma MEM, 10% FBS, and NEAA
Daudi lymphoblast human blood from a lymphoma patient RPMI-1640, 10% FBS
GH1, GH3 epithelial rat pituitary tumor F-10, 15% horse serum, and 2.5% FBS
* BME: Basal Medium Eagle; DMEM: Dulbecco’s Modified Eagle Medium; FBS: Fetal Bovine Serum; GMEM: Glasgow Minimum Essential Medium;
IMDM: Iscove’s Modified Dulbecco’s Medium; MEM: Minimum Essential Medium; NEAA: Non-Essential Amino Acids Solution.

Mammalian Cell Culture, continued
Cell Line Cell Type Species Tissue Medium*
H9 lymphoblast human T-cell lymphoma RPMI-1640, 20% FBS
HaK epithelial hamster kidney BME, 10% calf serum
HCT-15 epithelial human colorectal adenocarcinoma RPMI-1640, 10% FBS
HeLa epithelial human cervix carcinoma MEM, 10% FBS, and NEAA (in suspension, S-MEM)
HEp-2 epithelial human larynx carcinoma MEM, 10% FBS
HL-60 lymphoblast human promyeolocytic leukemia RPMI-1640, 20% FBS
HT-1080 epithelial human fibrosarcoma MEM, 10% HI FBS, and NEAA
HT-29 epithelial human colon adenocarcinoma McCoy's 5A, 10% FBS
HUVEC endothelial human umbilical cord F-12K, 10% FBS, and 100 μg/mL heparin
I-10 epithelial mouse testicular tumor F-10, 15% horse serum, and 2.5% FBS
IM-9 lymphoblast human marrow from myeloma patient RPMI-1640, 10% FBS
JEG-2 epithelial human choriocarcinoma MEM, 10% FBS
Jensen fibroblast rat sarcoma McCoy's 5A, 5% FBS
Jurkat lyphoblast human lymphoma RPMI-1640, 10% FBS
K-562 lymphoblast human myelogenous leukemia RPMI-1640, 10% FBS
KB epithelial human oral carcinoma MEM, 10% FBS, and NEAA
marrow from erythroleukemia

KG-1 myeloblast human IMDM, 20% FBS
patient
L2 epithelial rat lung F-12K, 10%FBS
LLC-WRC 256 epithelial rat carcinoma Medium 199, 5% horse serum
McCoy fibroblast mouse unknown MEM, 10% FBS
MCF7 epithelial human breast adenocarcinoma MEM, 10% FBS, NEAA, and 10 μg/mL insulin
WI-38 epithelial human embryonic lung BME, 10% FBS
WISH epithelial human amnion BME, 10% FBS
XC epithelial rat sarcoma MEM, 10% FBS, and NEAA
Y-1 epithelial mouse tumor of adrenal F-10, 15% horse serum, and 2.5% FBS
Insect Cell Culture
fall army worm TNM-FH and 10% FBS, or Sf-900 II SFM (serum-
Sf9, Sf21 pupal ovary
(Spodoptera frugiperda) free), or Sf-900™ III SFM (serum-free)
High Five™ cabbage looper TNM-FH and 10% FBS, or Express Five® SFM
ovary
(BTI-TN-5B1-4) (Trichoplusia ni) (serum-free)
Schneider 2 (S2), fruit fly Schneider’s Drosophila medium and 10% heat-
D.Mel-2 (Drosophila melanogaster) inactivated FBS
* BME: Basal Medium Eagle; DMEM: Dulbecco’s Modified Eagle Medium; FBS: Fetal Bovine Serum; GMEM: Glasgow Minimum Essential Medium;
IMDM: Iscove’s Modified Dulbecco’s Medium; MEM: Minimum Essential Medium; NEAA: Non-Essential Amino Acids Solution;
TNM-FH: Trichoplusia ni Medium-Formulation Hink (i.e., Grace’s Insect Medium, Supplemented).
29
Dissociating adherent
cells The first step in subculturing adherent cells is to detach them from the surface of the
culture vessel by enzymatic or mechanical means. The table below lists the various cell
dissociation procedures.
Procedure Dissociation Agent Applications
Gentle shaking or rocking of

Loosely adherent cells, mitotic
Shake-off culture vessel, or vigorous
cells
pipetting.
Cell lines sensitive to proteases;

Scraping Cell scraper
may damage some cells
Trypsin Strongly adherent cells
High density cultures, cultures

Trypsin + collagenase that have formed muliple layers,
especially fibroblasts
Detaching epidermal cells as

Enzymatic confluent, intact sheets from the
dissociation Dispase
surface of culture dishes without
dissociating the cells
Strongly adherent cells; direct

™ substitute for trypsin; applications
TrypLE dissociation enzyme
that require animal origin-free
reagents
TrypLE™ dissociation
enzymes TrypLE™ Express and TrypLE™ Select are microbially produced cell dissociation
enzymes with similar kinetics and cleavage specificities to trypsin. Although TrypLE™
enzymes can directly substitute trypsin in dissociation procedures without a need for
protocol changes, we recommend that you initially optimize the incubation time for
dissociation for best results. Because TrypLE™ enzymes are recombinant fungal trypsin-
like proteases, they are ideal for applications that require animal origin-free reagents.
The table below compares TrypLE™ Express and TrypLE™ Select to trypsin.
TrypLE™ Express and TrypLE™ Select Trypsin
Completely free of animal- and human- Porcine- or bovine-derived

derived components
Stable at room temperature for at least six Not stable at room temperature.
months.
Not inhibited by serum Inhibited by serum
Does not require trypsin inactivators Requires trypsin inactivators

Subculturing Adherent Cells

The following protocol describes a general procedure for subculturing adherent
mammalian cells in culture. Note that the procedure for passaging insect cells differs
from that for mammalian cells on several crucial steps. For more information, refer to
Notes on Subculturing Insect Cells, next page.
For passaging your own cell line, we recommend that you closely follow the
instructions provided with each product you are using in your experiments. The
consequences of deviating from the culture conditions required for a particular cell type
can range from the expression of aberrant phenotypes to a complete failure of the cell
culture.
Materials needed • Culture vessels containing your adherent cells

• Tissue-culture treated flasks, plates or dishes
• Complete growth medium, pre-warmed to 37°C
• Disposable, sterile 15-mL tubes
• 37°C incubator with humidified atmosphere of 5% CO2
• Balanced salt solution such as Dulbecco’s Phosphate Buffered Saline (DPBS),
containing no calcium, magnesium, or phenol red
• Dissociation reagent such as trypsin or TrypLE™ Express, without phenol red
• Reagents and equipment to determine viable and total cell counts (e.g., Countess® II
Automated Cell Counter)
Protocol for passaging

adherent cells All solutions and equipment that come in contact with the cells must be sterile.
Always use proper sterile technique and work in a laminar flow hood.
1. Remove and discard the spent cell culture media from the culture vessel.
2. Wash cells using a balanced salt solution without calcium and magnesium
(approximately 2 mL per 10 cm2 culture surface area). Gently add wash solution to the
side of the vessel opposite the attached cell layer to avoid disturbing the cell layer, and
rock the vessel back and forth several times.
Note: The wash step removes any traces of serum, calcium, and magnesium that would
inhibit the action of the dissociation reagent.
3. Remove and discard the wash solution from the culture vessel
4. Add the pre-warmed dissociation reagent such as trypsin or TrypLE™ to the side of
the flask; use enough reagent to cover the cell layer (approximately 0.5 mL per 10 cm2).
Gently rock the container to get complete coverage of the cell layer.
5. Incubate the culture vessel at room temperature for approximately 2 minutes. Note that
the actual incubation time varies with the cell line used.
31
6. Observe the cells under the microscope for detachment. If cells are less than 90%
detached, increase the incubation time a few more minutes, checking for dissociation
every 30 seconds. You may also tap the vessel to expedite cell detachment.
7. When ≥ 90% of the cells have detached, tilt the vessel for a minimal length of time to
allow the cells to drain. Add the equivalent of 2 volumes (twice the volume used for the
dissociation reagent) of pre-warmed complete growth medium. Disperse the medium
by pipetting over the cell layer surface several times.
8. Transfer the cells to a 15-mL conical tube and centrifuge then at 200 × g for 5 to
10 minutes. Note that the centrifuge speed and time vary based on the cell type.
9. Resuspend the cell pellet in a minimal volume of pre-warmed complete growth

medium and remove a sample for counting.
10. Determine the total number of cells and percent viability using a hemocytometer, cell
counter and Trypan Blue exclusion, or the Countess® II Automated Cell Counter. If
necessary, add growth media to the cells to achieve the desired cell concentration and
recount the cells.
11. Dilute cell suspension to the seeding density recommended for the cell line, and
pipet the appropriate volume into new cell culture vessels, and return the cells to the
incubator.
Note: If using culture flasks, loosen the caps before placing them in the incubator to
allow proper gas exchange unless you are using vented flasks with gas-permeable caps.
Notes on subculturing
adherent insect cells While the general procedure for subculturing insect cells follows the same steps as
mammalian cells, some key requirements of these culture systems are different. For best
results, always follow the instructions provided with each product you are using in
your experiments.
• Passage insect cells at log phase. However, if your insect cells are strongly adherent,
you may passage them at confluency or slightly after when they are starting to pull
away from the bottom of the flask. Cells will be easier to dislodge.
• Densities lower than 20% confluency inhibit growth. The healthiest cells are those
taken from log phase cultures.
• CO2 exchange is not recommended for insect cell culture.
• Maintain insect cells at 27°C in a non-humidified environment. Cells can be
maintained at room temperature on the bench top if protected from light or in a
drawer. However, a 27°C controlled environment is recommended.
• Use media specifically formulated for insect cell growth.
• Insect cells attach very tightly to substrates under serum-free conditions and require
additional effort to detach. To dislodge the cells, you may need to give the flask one
quick shake using a wrist-snapping motion. To avoid contamination, always tighten
the cap before this procedure.
Caution: We do not recommend shaking the flask vigorously, because it may result in
damage to the cells.

Subculturing Suspension Cells

The following protocols describe general procedures for subculturing mammalian
cells in suspension culture. Note that the procedure for passaging insect cells differs
from that for mammalian cells on several crucial steps. For more information, refer to
Notes on Subculturing Insect Cells, page 36.
For passaging your own cell line, we recommend that you closely follow the
instructions provided with each product you are using in your experiments. The
consequences of deviating from the culture conditions required for a particular cell type
can range from the expression of aberrant phenotypes to a complete failure of the cell
culture.
Passaging suspension
cultures Subculturing suspension cells is somewhat less complicated than passaging adherent
cells. Because the cells are already suspended in growth medium, there is no need to
treat them enzymatically to detach them from the surface of the culture vessel, and the
whole process is faster and less traumatic for the cells. Replacement of growth medium
is not carried out in suspension cultures; instead, the cells are maintained by feeding
them every 2 to 3 days until they reach confluency. This can be done by directly diluting
the cells in the culture flask and continue expanding them, or by withdrawing a portion
of the cells from the culture flask and diluting the remaining cells down to a seeding
density appropriate for the cell line. Usually, the lag period following the passaging is
shorter than that observed with adherent cultures.
Suspension culture
vessels Suspension cultures can be maintained in sterile culture flasks that are not tissue-
culture treated; however, spinner flasks (i.e., stirrer bottles) specifically designed for
suspension cell culture allow for superior gas exchange and permit higher volumes
of cells to be cultured. Roller bottles rotating on a rack may also be used to agitate
suspension cultures.
Spinner flasks have two basic designs; the medium is agitated (i.e., stirred) by a
hanging stir-bar assembly or with a vertical impeller. The vertical impeller provides
better aeration. The total culture volume in a spinner flask should not exceed half of
the indicated volume of the spinner for proper aeration (e.g., a 500 mL spinner should
never contain more than 250 mL of culture).

33
Materials needed • Culture vessels containing your suspension cells

• Shaker flasks without baffles or spinner bottles (see Suspension Culture Vessels,
previous page)
• 37°C incubator with humidified atmosphere of 5% CO2
• Magnetic stir plate (if using spinner flasks), roller rack (if using roller bottles), or
shaking platform (if using conventional culture flasks or petri dishes)
Protocol for passaging

suspension cells All solutions and equipment that come in contact with the cells must be sterile.
Always use proper sterile technique and work in a laminar flow hood. Subculture cells
when they are in log-phase growth before they reach confluency. When they reach
confluency, cells in suspension clump together and the medium appears turbid when
the culture flask is swirled. The maximum recommended cell density before passaging
varies with cell lines; refer to the cell-specific product insert or manual for details.
Cells grown in shaker flasks

The following protocol describes a general procedure for passaging mammalian cells
grown in suspension culture using shaker flasks in a shaking incubator. For detailed
protocols, always refer to the cell-specific product insert.
Note: Make sure that the shaker flask does not have baffles (i.e., the indents at the
bottom of the flask designed to provide agitation), because they ruin the shaking
rhythm.
1. When the cells are ready for passaging (i.e., log-phase growth before they reach
confluency), remove the flask from the shaking incubator, and take a small sample
from the culture flask using a sterile pipette. If cells have settled down before taking the
sample, swirl the flask to evenly distribute the cells in the medium.
2. From the sample, determine the total number of cells and percent viability using the
Countess® II Automated Cell Counter or a hemocytometer, cell counter, and Trypan
Blue exclusion.
3. Calculate the volume of media that you need to add to dilute the culture down to the
recommended seeding density.
4. Aseptically add the appropriate volume of pre-warmed growth medium into the
culture flask. You may split the culture to multiple flasks if needed.
5. Loosen the caps of the culture flasks one full turn to allow for proper gas exchange (or
use a gas-permeable cap), and return the flasks to the shaking incubator. The shaking
speed depends on the cell line.
Note: To minimize the accumulation of cell debris and metabolic waste by-products
in shaker cultures, gently centrifuge the cell suspension at 100 × g for 5 to 10 minutes,
and resuspend the cell pellet in fresh growth medium once every three weeks (or as
needed).

Cells grown in spinner flasks

The following protocol describes a general procedure for passaging mammalian cells in
suspension grown using spinner flasks. For detailed protocols, always refer to the cell-
specific product insert.
Note that cells are sensitive to physical shearing. Ensure that impeller mechanisms
rotate freely and do not contact vessel walls or the base. The top of the paddles should
be slightly above the medium to ensure adequate aeration to the culture. Adjust the
spinner mechanism so that paddles clear the sides and the bottom of the vessel. The
table below lists the minimum volumes of media needed for different spinner flask
sizes.
Size of Spinner Flask Minimum Media Volume
100 mL 30 mL
250 mL 80 mL
500 mL 200 mL
We do not recommend initiating a spinner culture into a spinner flask larger than
500 mL. We suggest scaling up from smaller spinners that have already been
established.
1. When the cells are ready for passaging (i.e., log-phase growth before they reach
confluency), remove the flask from the shaking incubator, and take a small sample
from the culture flask using a sterile pipette. If cells have settled down before taking the
sample, swirl the to evenly distribute the cells in the medium.
2. From the sample, determine the total number of cells and percent viability using the
Countess® II Automated Cell Counter or a hemocytometer, cell counter and Trypan Blue
exclusion.
3. Calculate the volume of media that you need to add to dilute the culture down to the
recommended seeding density.
4. Aseptically add the appropriate volume of pre-warmed growth medium into the
culture flask. You may split the culture to multiple flasks if needed.
5. Loosen the side arm caps of the spinner flasks one full turn to allow for proper gas
exchange, and return the flasks to the incubator. The spinner speed depends on the
cell line and the impeller type. Make sure that the spinner speed is kept within the
recommended values to avoid damage to the cells from shear stress.
Note: To minimize the accumulation of cell debris and metabolic waste by-products
in spinner cultures, gently centrifuge the cell suspension at 100 × g for 5 to 10 minutes,
and resuspend the cell pellet in fresh growth medium once every three weeks (or as
needed).
35
Notes on subculturing
suspension insect cells While the general procedure for subculturing insect cells follows the same steps as
mammalian cells, some key requirements of these culture systems are different. For
best results, always follow the instructions provided with the insect cell lines you are
using in your experiments.
• It is not necessary to change medium when you are culturing cells in suspension.
Regular subculturing requires the removal of cell suspension and the addition of
medium sufficient to dilute culture to the appropriate density (refer to the cell-
specific product insert). Adding fresh medium is sufficient to replenish cell nutrients.
• CO2 exchange is not recommended for insect cell culture.
• Maintain insect cells at 27°C in a non-humidified environment. Cells can be
maintained at room temperature on the bench top or in a drawer, however, a 27°C
controlled environment is recommended.
• Use media specifically formulated for insect cell growth.
• Use a surfactant to decrease shearing. 0.1% Pluronic® F-68 is recommended for
spinner insect cultures. Pluronic® F-68 is a surfactant that decreases cell membrane
shearing due to impeller forces.
Note: Sf-900 II SFM and Express Five® SFM already contain surfactants.
• Certain insect cell lines may require adaptation to suspension culture. For more
information, refer to the cell-line specific product insert or manual.

Freezing Cells
Cryopreservation Cell lines in continuous culture are prone to genetic drift, finite cell lines are fated for
senescence, all cell cultures are susceptible to microbial contamination, and even the
best-run laboratories can experience equipment failure. Because an established cell line
is a valuable resource and its replacement is expensive and time consuming, it is vitally
important that they are frozen down and preserved for long-term storage.
As soon as a small surplus of cells becomes available from subculturing, they should be
frozen as a seed stock, protected, and not be made available for general laboratory use.
Working stocks can be prepared and replenished from frozen seed stocks. If the seed
stocks become depleted, cryopreserved working stocks can then serve as a source for
preparing a fresh seed stock with a minimum increase in generation number from the
initial freezing.
The best method for cryopreserving cultured cells is storing them in liquid nitrogen in
complete medium in the presence of a cryoprotective agent such as dimethylsulfoxide
(DMSO). Cryoprotective agents reduce the freezing point of the medium and also
allow a slower cooling rate, greatly reducing the risk of ice crystal formation, which can
damage cells and cause cell death.
Note: DMSO is known to facilitate the entry of organic molecules into tissues. Handle
reagents containing DMSO using equipment and practices appropriate for the hazards
posed by such materials. Dispose of the reagents in compliance with local regulations.
Guidelines for
cryopreservation Following the guidelines below is essential for cryopreserving your cell lines for future
use. As with other cell culture procedures, we recommend that you closely follow the
instructions provided with your cell line for best results.
• Freeze your cultured cells at a high concentration and at as low a passage number as
possible. Make sure that the cells are at least 90% viable before freezing. Note that the
optimal freezing conditions depend on the cell line in use.
• Freeze the cells slowly by reducing the temperature at approximately 1°C per minute
using a controlled rate cryo-freezer or a cryo-freezing container (e.g., “Mr. Frosty,”
available from NALGENE® labware)
• Always use the recommended freezing medium. The freezing medium should
contain a cryoprotective agent such as DMSO or glycerol (see What is Subculture?,
page 26).
• Store the frozen cell below –70°C; frozen cells begin to deteriorate above –50°C.
• Always use sterile cryovials for storing frozen cells. Cryovials containing the frozen
cells may be stored immersed in liquid nitrogen or in the gas phase above the liquid
nitrogen (see Safety Note, page 38).
• Always wear personal protective equipment.
• All solutions and equipment that come in contact with the cells must be sterile.
Always use proper sterile technique and work in a laminar flow hood.
37
Safety note
Biohazardous materials must be stored in the gas phase above the liquid nitrogen.
Storing the sealed cryovials in the gas phase eliminates the risk of explosion. If you are
using liquid-phase storage, be aware of the explosion hazard with both glass and plastic
cryovials, and always wear a face shield or goggles.
Freezing medium Always use the recommended freezing medium for cryopreserving your cells. The
freezing medium should contain a cryoprotective agent such as DMSO or glycerol.
You may also use a specially formulated complete cryopreservation medium such
as Recovery™ Cell Culture Freezing Medium or Synth‑a-Freeze® Cryopreservation
Medium.
Recovery™ Cell Culture Freezing Medium is a ready-to-use complete cryopreservation

medium for mammalian cell cultures, containing an optimized ratio of fetal bovine
serum to bovine serum for improved cell viability and cell recovery after thawing.
Synth-a-Freeze® Cryopreservation Medium is a chemically defined, protein-

free, sterile cryopreservation medium containing 10% DMSO that is suitable for
the cryopreservation of many stem and primary cell types, with the exception of
melanocytes.
Materials needed • Culture vessels containing cultured cells in log-phase of growth

• Complete growth medium
• Cryoprotective agent such as DMSO (use a bottle set aside for cell culture; open
only in a laminar flow hood) or a freezing medium such as Synth-a-Freeze®
Cryopreservation Medium or Recovery™ Cell Culture Freezing Medium
• Disposable, sterile 15-mL or 50-mL conical tubes
• Sterile cryogenic storage vials (i.e., cryovials)
• Controlled rate freezing apparatus or isopropanol chamber
• Liquid nitrogen storage container
For freezing adherent cells, in addition to the above materials, you need:
• Balanced salt solution such as Dulbecco’s Phosphate Buffered Saline (DPBS),

containing no calcium, magnesium, or phenol red
• Dissociation reagent such as trypsin or TrypLE™ Express, without phenol red

Cryopreserving cultured
cells The following protocol describes a general procedure for cryopreserving cultured cells.
For detailed protocols, always refer to the cell-specific product insert.
1. Prepare freezing medium and store at 2°C to 8°C until use. Note that the appropriate
freezing medium depends on the cell line.
2. For adherent cells, gently detach cells from the tissue culture vessel following the
procedure used during the subculture. Resuspend the cells in complete medium
required for that cell type.
3. Determine the total number of cells and percent viability using a hemocytometer,
cell counter and Trypan Blue exclusion, or the Countess® II Automated Cell Counter.
According to the desired viable cell density, calculate the required volume of freezing
medium.
4. Centrifuge the cell suspension at approximately 100–200 × g for 5 to 10 minutes

Aseptically decant supernatant without disturbing the cell pellet.
Note: Centrifugation speed and duration varies depending on the cell type.
5. Resuspend the cell pellet in cold freezing medium at the recommended viable cell
density for the specific cell type.
6. Dispense aliquots of the cell suspension into cryogenic storage vials. As you aliquot
them, frequently and gently mix the cells to maintain a homogeneous cell suspension.
7. Freeze the cells in a controlled rate freezing apparatus, decreasing the temperature
approximately 1°C per minute. Alternatively, place the cyrovials containing the cells in
an isopropanol chamber and store them at –80°C overnight.
8. Transfer frozen cells to liquid nitrogen, and store them in the gas phase above the liquid
nitrogen.
39
Thawing Frozen Cells

Guidelines for thawing The thawing procedure is stressful to frozen cells, and using good technique and
working quickly ensures that a high proportion of the cells survive the procedure.
As with other cell culture procedures, we recommend that you closely follow the
instructions provided with your cells and other reagents for best results.
• Thaw frozen cells rapidly (< 1 minute) in a 37°C water bath.

• Dilute the thawed cells slowly, using pre-warmed growth medium.
• Plate thawed cells at high density to optimize recovery.
• Always use proper aseptic technique and work in a laminar flow hood.
• Always wear personal protective equipment, including a face mask or goggles.
Cryovials stored in liquid-phase present a risk of explosion when thawed.
• Some freezing media contain DMSO, which is known to facilitate the entry of organic
molecules into tissues. Handle reagents containing DMSO using equipment and
practices appropriate for the hazards posed by such materials.
Materials needed • Cryovial containing frozen cells

• Disposable, sterile centrifuge tubes
• Water bath at 37°C
• 70% ethanol
• Tissue-culture treated flasks, plates, or dishes
Thawing frozen cells The following protocol describes a general procedure for thawing cryopreserved cells.
For detailed protocols, always refer to the cell-specific product insert.
1. Remove the cryovial containing the frozen cells from liquid nitrogen storage and
immediately place it into a 37°C water bath.
2. Quickly thaw the cells (< 1 minute) by gently swirling the vial in the 37°C water bath
until there is just a small bit of ice left in the vial.
3. Transfer the vial it into a laminar flow hood. Before opening, wipe the outside of the
vial with 70% ethanol.
4. Transfer the thawed cells dropwise into the centrifuge tube containing the desired
amount of pre-warmed complete growth medium appropriate for your cell line.
5. Centrifuge the cell suspension at approximately 200 × g for 5–10 minutes. The actual
centrifugation speed and duration varies depending on the cell type.
6. After the centrifugation, check the clarity of supernatant and visibility of a complete
pellet. Aseptically decant the supernatant without disturbing the cell pellet.
7. Gently resuspend the cells in complete growth medium, and transfer them into the
appropriate culture vessel and into the recommended culture environment.
Note: The appropriate flask size depends on the number of cells frozen in the cryovial,
and the culture environment varies based on the cell and media type.

5. Transfection Basics
This chapter provides an overview of transfection, including general information on
various transfection technologies and selecting the appropriate transfection method for
your cell line and experimental needs. Guidelines for transfection of cells with DNA
and RNA, considerations for successful transfection experiments, and experimental
workflows are provided in the Transfection Methods chapter, starting on page 69.
Introduction to Transfection
What is transfection? Broadly defined, transfection is the process of artificially introducing nucleic acids
(DNA or RNA) into cells, utilizing means other than viral infection. Such introductions
of foreign nucleic acid using various chemical, biological, or physical methods can
result in a change of the properties of the cell, allowing the study of gene function and
protein expression in the context of the cell.
In transfection, the introduced nucleic acid may exist in the cells transiently, such that
it is only expressed for a limited period of time and does not replicate, or it may be
stable and integrate into the genome of the recipient, replicating when the host genome
replicates (see Types of Transfection, page 43).
Terminology The terminology used for various gene delivery systems has evolved to keep pace with
technological advances in the field and further refined to distinguish various methods
and cell types.
Transfection
Transfection commonly refers to the introduction of nucleic acids into eukaryotic
cells, or more specifically, into animal cells. Classically, the term transfection was
used to denote the uptake of viral nucleic acid from a prokaryote‑infecting virus
or bacteriophage, resulting in an infection and the production of mature virus
particles. However, the term has acquired its present meaning to include any artificial
introduction of foreign nucleic acid into a cell.
Transformation
Transformation is often used to describe non-viral DNA transfer in bacteria,
non‑animal eukaryotic cells, and plant cells. However, transformation also refers to a
particular event or a series of events that results in a permanent change in an animal
cell’s phenotype, and implies genetic instability and a progression to a cancerous state.
Although transformation in this sense can arise from infection with a transforming
virus or from gene transfection, it can also arise spontaneously or following external
stressors such as ionizing radiation or chemical mutagens. As such, the term should be
avoided for animal cells when describing introduction of exogenous genetic material.
Transduction
Transduction is used to describe virus-mediated DNA transfer. However, the term
transfection is also used to refer to infecting a cell specifically with viral nucleic acid
that is isolated either from a eukaryote virus or from a bacteriophage.

Part 5. Transfection Basics
Applications The two main purposes of transfection are to produce recombinant proteins, or to
specifically enhance or inhibit gene expression in transfected cells. As such, transfection
is a powerful analytical tool for the study of the function and regulation of genes or
gene products, for the production of transgenic organisms, and as a method for gene
therapy.
Gene expression
Transfection is most commonly performed to express a protein of interest in cultured
cells (or an animal model) through the use of a plasmid vector or mRNA. Expression
of the protein in eukaryotic cells allows the recombinant protein to be produced with
proper folding and post-translational modifications required for its function. Further,
introducing proteins with readily detectable markers and other modifications into cells
allows the study of promoter and enhancer sequences or protein:protein interactions.
In addition, transfection can be used in various forms of bioproduction depending upon

the transfection strategy. For example, delivery of reprogramming transcription factors
enables the generation of induced pluripotent stem cell (iPSC). Stable transfection,
on the other hand, provides the means for the bioproduction of various therapeutic
molecules.
Gene inhibition
Another frequent use of transfection is in inhibiting the expression of specific proteins
through RNA interference (RNAi). In mammalian cells, RNAi occurs through
endogenously expressed non-coding RNA in the form of microRNAs (miRNAs),
which are derived from a double-stranded RNA (dsRNA) precursor. The precursor is
processed to a mature miRNA that becomes part of a RNA-induced silencing complex
(RISC), which acts to inhibit translation of complementary target mRNAs.
Vector-based systems express miRNA precursors or short hairpin RNA (shRNA)

precursors that are processed by endogenous machinery to produce miRNAs or
shRNAs, respectively, which then act to inhibit gene expression. These systems allow
stable transfection of recombinant constructs, and can permit inducible expression of
precursor molecules.
Chemically synthesized short/small interfering RNAs (siRNAs) can also be

incorporated into a RISC and induce gene silencing by targeting complementary mRNA
for degradation. Modifications to siRNAs help to prevent off-target effects, and also to
ensure that the active strand of the dsRNA is loaded into the RISC.

Types of Transfection
There are a number of biological, chemical, and physical methods for introducing
nucleic acids into cells. Not all of these methods can be applied to all types of cells and
experimental applications, and there is a wide variation amongst them with respect
to transfection efficiency, cell toxicity, effects on normal physiology, and level of gene
expression. However, all of the transfection strategies can be broadly classified into
two general types based on whether the introduced nucleic acid exists in the cell for
a limited period of time (transient transfection) or whether it persists in the cells
long‑term and is passed to the progeny of the transfected cell (stable transfection).
Transient transfection In transient transfection, the introduced nucleic acid exists in the cell only for a limited
period of time and is not integrated into the genome. As such, transiently transfected
genetic material is not passed from generation to generation during cell division, and it
can be lost by environmental factors or diluted out during cell division. However, the
high copy number of the transfected genetic material leads to high levels of expressed
protein within the period that it exists in the cell.
Depending on the construct used, transiently expressed transgene can generally be

detected for 1 to 7 days, but transiently transfected cells are typically harvested 24 to
96 hours post-transfection. Analysis of gene products may require isolation of RNA
or protein for enzymatic activity assays or immunoassays. The optimal time interval
depends on the cell type, research goals, and specific expression characteristics of
the introduced gene, as well as the time it takes for the reporter to reach steady state.
However, within a few days most of the foreign DNA is degraded by nucleases or
diluted by cell division; after a week, its presence is no longer detected.
Transient transfection is most efficient when supercoiled plasmid DNA is used,

presumably due to its more efficient uptake by the cell. siRNAs, miRNAs, mRNAs,
and even proteins can be also used for transient transfection, but as with plasmid
DNA, these macromolecules need to of high quality and relatively pure (see Factors
Influencing Transfection Efficiency, page 69). While transfected DNA is translocated
into the nucleus for transcription, transfected RNA remains in the cytosol, where it is
expressed within minutes after transfection (mRNA) or bound to mRNA to silence the
expression of a target gene (siRNA and miRNA) (see Guidelines for RNA Transfection,
page 90).
Stable transfection In stable transfection, foreign DNA is either integrated into the cellular genome or
maintained as an episomal plasmid. Unlike transient transfection, stable transfection
allows the long-term maintenance of the exogenous DNA in the transfected cell and
its progeny. As such, stable transfection can provide persistent expression of the
introduced gene through multiple generations, which can be useful for production of
recombinant proteins and analysis of downstream or long-term effects of exogenous
DNA expression. However, usually a single or a few copies of the exogenous DNA is
integrated into the genome of the stably transfected cell. For this reason, the expression
level of stably transfected genes tend to be lower than that of transiently transfected
genes.
43
Because stable integration of foreign DNA into the genome is a relatively rare event,
successful stable transfection requires both effective DNA delivery and a way to select
cells that have acquired the DNA. One of the most reliable ways to select cells that
stably express transfected DNA is to include a selectable marker in the DNA construct
used for transfection and then apply the appropriate selective pressure to the cells after
a short recovery period (see Selective Screening, page 62).
Frequently used selectable markers are genes that confer resistance to various selection
drugs or genes that compensate for an essential gene that is defective in the cell line
to be transfected. When cultured in selective medium, cells that were not transfected
or were transiently transfected eventually die, and those that express the antibiotic
resistance gene at sufficient levels or those that can compensate for the defect in the
essential gene survive.
Alternatively, phenotypical or morphological changes in the transfected cells can be

used as a screenable trait in certain cases. For example, mouse CI127 cells transfected
with vectors derived from bovine papilloma virus produce a morphological change
(Sarver et al. 1981).
Although linear DNA results in lower DNA uptake by the cells relative to supercoiled
DNA, it yields optimal integration of DNA into the host genome (see Factors
Influencing Transfection Efficiency, page 69). As a rule, stable transfection is limited
to DNA vectors, but siRNA and miRNA may be stably introduced into cells when
they are delivered as short hairpin transcripts made from a selectable DNA vector (see
Vector‑mediated RNAi, page 89). However, RNA molecules by themselves cannot be
used for stable transfection.
Choosing a transfection
strategy Deciding whether you need transient or stable transfection depends on the time frame
and ultimate goal of the experiment you wish to conduct. Transiently transfected
cells are typically harvested 24–96 hours post-transfection and are often used for
studying the effects of short-term expression of genes or gene products, performing
RNA interference (RNAi)‑mediated gene silencing, or rapidly producing recombinant
proteins on a small scale. Transient transfection with mRNA can deliver even
more rapid results; because mRNA is expressed in the cytosol without the need for
translocation to the nucleus and the transcription process, it is possible for transfected
mRNA to be expressed within minutes after transfection in some systems.
In contrast, stable transfection is more useful when long-term gene expression is

required or when transfected cells need to be used over many experiments. Because
integration of a DNA vector into the chromosome is a rare event, stable transfection of
cells is a more laborious and challenging process, which requires selective screening
and clonal isolation. As such, it is normally reserved for large-scale protein production,
longer-term pharmacology studies, gene therapy, or research on the mechanisms of
long-term genetic regulation.

Although transient transfection of mammalian cells has been employed for the
production of recombinant proteins with proper folding and post-translational
modifications (which are not available when expressing recombinant proteins in
bacterial cells) since the invention of transfection reagents, the ability to express
milligram-to-gram amounts of recombinant protein has relied mainly on the creation
of stable cell lines. More recently, large volume transient transfection of HEK293
and CHO cells adapted to suspension culture has addressed the need to obtain high
amounts of recombinant protein without having to resort to the laborious process of
stable cell line development. Recombinant protein expression by transient transfection
enables researchers to produce, starting from the vector of interest and suspension-
adapted CHO or HEK293 cells, milligram-per-liter quantities of correctly folded and
glycosylated recombinant proteins in three to seven days.
A major advancement in transient expression technology for rapid and ultra high-yield
protein production in mammalian cells is the Expi293™ Expression System, which is
based on the high-density culture of Expi293F™ cells in Expi293™ Expression Medium
and transfection using the cationic lipid-based ExpiFectamine™ 293 transfection reagent
in combination with optimized transfection enhancers. All components work in concert
to generate 2- to 10-fold higher protein yields than conventional culture systems such
as the FreeStyle™ 293 Expression System, achieving expression levels of greater than
1 g/L for IgG and non-IgG proteins. For more information on the Expi293™ Expression
System, go to www.lifetechnologies.com/expi293.
Clinical biotherapeutics are frequently generated using stable, high-expression

transfectants, because they provide batch-to-batch consistency and low cost at
extremely large-scales. However, in many drug discovery applications, it is beneficial
to screen protein constructs quickly using transient transfection methods, which allow
simultaneous evaluation of various candidate molecules in less than one week. In
many instances, transient transfections are performed in parallel while more resource-
intensive stable cell lines are under development, which can take more than three
months to accomplish.
Transient Transfection Stable Transfection
Transfected DNA is not integrated into the Transfected DNA integrates into the
genome, but remains in the nucleus. genome.
Transfected genetic material is not passed Transfected genetic material is carried

onto the progeny; genetic alteration is not stably from generation to generation;
permanent. genetic alteration is permanent.
Does not require selection. Requires selective screening for the

isolation of stable transfectants.
Both DNA vectors and RNA can be used for Only DNA vectors can be used for stable
transient transfection. transfection; RNA by itself cannot be stably
introduced into cells.
High copy number of transfected genetic Single or low copy number of stably
material results in high level of protein integrated DNA results in lower level of
expression. protein expression.
Cells are typically harvested within 24–96 Requires 2–3 weeks of selection for the
hours of transfection. isolation of stably transfected colonies.
Generally not suitable for studies using Suitable for studies using vectors with
vectors with inducible promoters. inducible promoters.
45
Gene Delivery Technologies

The cell membrane consists of a phospholipid bilayer with embedded proteins
and carries a net negative charge. Thus, it presents an impenetrable barrier to large
molecules that, like the phosphate backbones of DNA and RNA, are also negatively
charged. To sneak nucleic acids through the cell membrane, researchers have developed
a number of techniques each using a different approach—from using chemicals and
carrier molecules that coat the nucleic acids to neutralize them to physical methods that
create transient pores in the membrane to introduce the DNA directly into the cell.
Transfection technologies available today can be broadly classified into three groups:
chemical methods that use carrier molecules to neutralize or impart a positive charge
to the negatively charged nucleic acids, biological methods that rely on genetically
engineered viruses to transfer non-viral genes into cells (also known as transduction),
and physical methods that directly deliver nucleic acids into the cytoplasm or
the nucleus of the cell. However, no one method can be applied to all cells and all
experiments. The ideal approach should be selected depending your cell type and
experimental needs, should have high transfection efficiency, low cell toxicity, and
minimal effects on normal physiology, and be easy to use and reproducible (Kim and
Eberwine, 2010).
Chemical gene delivery methods
Technology Advantages Disadvantages
• Fast and easy protocols • Optimization may be necessary—some cell lines are
• Commercially available with reproducible results sensitive to cationic lipids
• High efficiency and expression performance • Some cell lines are not readily transfected with cationic
lipids
• Applicable to a broad range of cell lines and
Cationic lipid- high-throughput screens • Presence of serum may interefere with complex
mediated delivery formation and lower transfection efficiency
• Can be used for delivering DNA, RNA, and proteins
• Absence of serum in the medium may increase
• No size limitation on the packaged nucleic acid
cytotoxicity
• Applicable to both transient and stable protein
production
• Can be used for in vivo delivery of nucleic acids
• Inexpensive and easily available • Requires careful preparation of reagents—CaPO4

• Applicable to both transient and stable protein solutions are sensitive to changes in pH, temperature,
production and buffer salt concentrations
Calcium phosphate • High efficiency (cell line dependent) • Reproducibility can be problematic
co-precipitation • Cytoxicity, especially in primary cells
• Does not work with RPMI due to high phosphate
concentration of the medium
• Not suited for in vivo gene transfer to whole animals
• Relatively simple technique • Chemical cytoxicity in some cell types

DEAE-dextran • Reproducible results • Limited to transient transfection
• Inexpensive • Low transfection efficiency, especially in primary cells
Delivery by other • Typically stable in serum and not temperature sensitive • Cytoxicity in some cell types
cationic polymers • High efficiency (cell line dependent) • Non-biodegradable (dendrimers)
(e.g., polybrene, • Reproducible results • Limited to transient transfection
PEI, dendrimers)

Biological gene delivery methods
• Highest efficiency amongst gene delivery methods • Cell lines to transfect must contain viral receptors
(80–90% transduction efficiency in primary cells) • Limited insert size (~10 kb for most viral vectors versus
• Works well with difficult to transfect cell types ~100 kb for non-viral vectors)
• Can be used for in vivo delivery of nucleic acids • Technically challenging and time consuming to
Viral delivery
• Can be used for making stable cell lines (retroviral generate recombinant viruses
vectors) or for transient expresssion (adenoviral • Present biosafety issues (activation of latent disease,
vectors) immunogenic reactions, cytotoxicity, insertional
mutagenesis, malignant transformation of cells)
Physical gene delivery methods
• Simple principle • Requires special instrument

• Reproducible results after optimization • Optimization of electrical pulse and field strength
• No need for vector parameters required
• Less dependent on cell type and condition • Significantly more manipulation of cells required
Electroporation
• Rapid transfection of large number of cells after • High toxicity levels may be observed
optimization • High mortality rate requires large number of cells
• Can irreversibly damage the membrane and lyse the
cells
• Less dependent on cell type and condition • Requires expensive instrument

• Can be used for in vivo delivery of nucleic acids • Causes physical damage to samples
Biolistic particle • Straightforward method with reliable results • High mortality rate requires large number of cells
delivery (particle • No limitation to the size and or number of genes that • Preparation of microparticles is required
bombardment) can be delivered • Relatively costly for research applications
• Primarily used for genetic vaccination and agricultural • Generally less efficient than electroporation or viral- or
applications lipid-mediated delivery
• Less dependent on cell type and condition • Requires expensive instrument

• Allows single-cell transfection • Technically demanding and very labor-intensive (one
Direct • Straightforward method with reliable results cell at a time)
microinjection • No limitation to the size and or number of genes that • Often causes cells deaths
can be delivered
• No need for vector
• Can be used for delivering DNA, RNA, proteins, • Requires expensive laser-microscope system
ions, dextrans, small molecules, and semiconductor • Requires cells to be attached
nanocrystals • Technically demanding
Laser-mediated • Can be applied to very small cells
transfection • Allows single-cell transfection or transfection of large
(phototransfection) number of cells at the same time
• No need for vector
• High efficiency
• Applicable to a broad range of cell lines
47
Cationic lipid-mediated
delivery Cationic lipid-mediated transfection is one of the most popular methods for introducing
foreign genetic material into cells. Although first generation of lipid-based transfection
reagents relied on artificial liposomes that could envelop nucleic acids and then fuse
with the cell membrane to deposit their cargo inside (Fraley et al., 1980), newer cationic
lipid-based reagents spontaneously form condensed nucleic acid-cationic lipid reagent
complexes via electrostatic interactions between the negatively charged nucleic acid
and the positively charged head group of the synthetic lipid reagent. These complexes
are believed to be taken up by the cell through endocytosis and then released in the
cytoplasm. Once in the cell, transfected DNA is translocated to the nucleus to be
expressed by a yet unknown mechanism, while RNA or antisense oligonucleotides
skip the translocation step and remain in the cytoplasm (see Cationic Lipid-Mediated
Transfection, page 58).
The advantages of cationic lipid-mediated transfection are the ability to transfect a

broad range of cell lines with high efficiency, its applicability to high-throughput
screens, and the ability to deliver DNA of all sizes, as well as RNA and proteins. In
addition, this method can be applied to both stable and transient expression, and
unlike other chemical methods, it can be used for in vivo transfer of DNA and RNA to
animals and humans. The main drawback of cationic lipid-mediated transfection is the
dependence of transfection efficiency on the cell type and culture conditions, requiring
the optimization of transfection conditions for each cell type and transfection reagent
(see Considerations for Cationic Lipid-Mediated Transfection, page 80).
Life Technologies™ offers a wide range of cationic lipid-mediated transfection reagents

for efficiently introducing DNA, RNA, siRNA, or oligonucleotides into a broad range
of cell types, including the Lipofectamine® 3000 reagent. The Lipofectamine® 3000
reagent leverages the most advanced lipid nanoparticle technology to enable superior
transfection efficiency and reproducible results in a broad spectrum of difficult-to-
transfect cell types with improved viability (www.lifetechnologies.com/3000). For more
information on selecting the appropriate transfection reagent for your application, see
Cationic lipid transfection reagents, page 56.
Figure 5.1 Cationic lipid-mediated transfection workflow.

Calcium phosphate
co‑precipitation Calcium phosphate co-precipitation has been a popular transfection method since its
introduction in the early 1970s (Graham and van der Eb, 1973) because the components
it requires are easily available and inexpensive. Furthermore, the technique is easy
to master, it is effective with many types of cultured cells, and it can be used for both
transient and stable transfection of a variety of cultured cell types. However, calcium
phosphate co-precipitation is prone to variability due to its sensitivity to slight changes
in pH, temperature, and buffer salt concentrations, and can be cytotoxic to many
types of cell cultures, especially of primary cells. In addition, it is unsuitable for in vivo
transfer of nucleic acids to whole animals, and it shows relatively poor transfection
efficiency compared to other chemical transfection methods such as lipid-mediated
transfection.
The principle of calcium phosphate co-precipitation involves mixing DNA with calcium
chloride in a buffered saline/phosphate solution to generate a calcium-phosphate–
DNA co-precipitate, which is then dispersed onto cultured cells. Calcium phosphate
facilitates the binding of the condensed DNA in the co-precipitate to the cell surface,
and the DNA enters the cell by endocytosis. Aeration of the phosphate buffer while
adding the DNA-calcium chloride solution helps to ensure that the precipitate that
forms is as fine as possible, which is important because clumped DNA will not adhere
to or enter the cell as efficiently.
Figure 5.2 Calcium-phosphate co-precipitation workflow.
49
DEAE-Dextran-mediated
delivery Diethylaminoethyl (DEAE)-dextran is a polycationic derivative of the carbohydrate
polymer dextran, and it is one of the first chemical reagents used to transfer nucleic
acids into cultured mammalian cells (Vaheri and Pagano, 1965). The cationic
DEAE‑dextran molecule tightly associates with the negatively charged backbone of the
nucleic acid, and the net positive charge of the resulting nucleic acid-DEAE‑dextran
complex allows it to adhere to the cell membrane and enter into the cytoplasm via
endocytosis or osmotic shock induced by DMSO or glycerol.
The advantages of DEAE-dextran method are its relative simplicity, reproducibility, and
low cost, while its disadvantages include cytotoxicity and low transfection efficiency for
a range of cell types (typically less than 10% in primary cells), as well as the requirement
for reduced serum media during the transfection procedure. In addition, this method is
limited to transient transfections, and is not suitable for generating stable cell lines.
Figure 5.3 DEAE-dextran-mediated transfection workflow.

Delivery by other cationic

polymers Other cationic polymers used for gene delivery include cationic peptides and their
derivatives (e.g., polylysine, polyornithine), linear or branched synthetic polymers
(e.g., polybrene, polyethyleneimine), polysaccharide-based delivery molecules (e.g.,
cyclodextrin, chitosan), natural polymers (e.g., histone, collagen), and activated and
non-activated dendrimers.
Cationic polymers differ from cationic lipids in that they do not contain a hydrophobic
moiety and are completely soluble in water. Although they differ dramatically in their
degree of transfection efficiency and cytotoxicity, all cationic polymers work in a similar
fashion by allowing the formation of nucleic acid-polymer complexes, which adhere
to the cell membrane through electrostatic interactions and are taken up by the cell via
endocytosis. The efficiency of uptake can be improved by conjugating cell-targeting
ligands or nuclear localization signals onto the polymer.
While cationic polymers can offer increased complex stability, more reproducible
results, and higher transfection efficiencies when compared to DEAE‑dextran,
their main limitations continue to be cytotoxicity and their limitation to transient
transfection studies. While higher molecular weight (MW) cationic polymers tend to
be non-biodegradable and more cytotoxic than lower MW polymers, they show higher
transfection efficiencies due to their increased polymer-to nucleic acid-charge ratio.
However, the higher toxicity of larger MW polymers can be reduced by biodegradable
cross-linking of small polymers into larger polymeric structures.
Figure 5.4 Cationic polymer-mediated transfection workflow.
51
Viral delivery For cell types not amenable to lipid-mediated transfection, viral vectors are often
employed. Virus-mediated transfection, also known as transduction, offers a means to
reach hard-to-transfect cell types for protein overexpression or knockdown, and it is the
most commonly used method in clinical research (Glover et al., 2005; Pfeifer and Verma,
2001). Adenoviral, oncoretroviral, and lentiviral vectors have been used extensively for
gene delivery in mammalian cell culture and in vivo. Other well-known examples for
viral gene transfer include baculovirus and vaccinia virus-based vectors. For the more
information on various viral delivery systems, see Virus-Mediated Gene Transfer,
page 58.
While viruses are the preferred system for gene delivery in clinical trials owing to
their high in vivo transfection efficiency and sustained gene expression due to their
integration into the host genome, they have a number of drawbacks including their
immunogenicity and cytotoxicity, technically challenging and laborious production
procedures for vectors, high costs due to biosafety requirements, low packaging
capacity (~10 kb for most viral vectors compared to ~100 kb for non-viral vectors), and
variability in the infectivity of viral vector preparations (Glover et al., 2005; Kim and
Eberwine, 2010; Vorburger and Hunt, 2002).
A typical transduction protocol involves engineering of the recombinant virus carrying

the transgene, amplification of recombinant viral particles in a packaging cell line,
purification and titration of amplified viral particles, and subsequent infection of the
cells of interest. While the achieved transduction efficiencies in primary cells and cell
lines are quite high (~90–100%), only cells carrying the viral-specific receptor can be
infected by the virus. It is also important to note that the packaging cell line used for
viral amplification needs to be transfected with a non-viral transfection method.
Figure 5.5 Viral delivery workflow.

Electroporation Electroporation is a physical transfection method that uses an electrical pulse to create
temporary pores in cell membranes through which substances like nucleic acids can
pass into cells. It is a highly efficient strategy for the introduction of foreign nucleic
acids into many cell types, including bacteria and mammalian cells.
Electroporation is based on a simple process. Host cells and selected molecules

are suspended in a conductive solution, and an electrical circuit is closed around
the mixture. An electrical pulse at an optimized voltage and only lasting a few
microseconds to a millisecond is discharged through the cell suspension. This disturbs
the phospholipid bilayer of the membrane and results in the formation of temporary
pores. The electric potential across the cell membrane simultaneously rises to allow
charged molecules like DNA to be driven across the membrane through the pores in a
manner similar to electrophoresis (Shigekawa and Dower, 1988).
The main advantage of electroporation is its applicability for transient and stable
transfection of all cell types. Furthermore, because electroporation is easy and rapid, it
is able to transfect a large number of cells in a short time once optimum electroporation
conditions are determined. The major drawback of electroporation is substantial cell
death caused by high voltage pulses and only partially successful membrane repair,
requiring the use of greater quantities of cells compared to chemical transfection
methods. While more modern instrumentation, such as the Neon® Transfection
System offered by Life Technologies™, overcome high cell mortality by distributing
the electrical pulse equally among the cells and maintaining a stable pH throughout
the electroporation chamber, optimization of pulse and field strength parameters is
still required to balance the electroporation efficiency and cell viability (see Neon®
Transfection System, page 60).
Figure 5.6 Electroporation workflow.
53
Other physical delivery

methods Physical gene delivery methods other than electroporation include biolistic particle
delivery, direct microinjection, and laser-mediated transfection. Although these
physical methods differ in the tools they employ, they all enable the direct transfer of
nucleic acids into the cytoplasm or the nucleus by membrane penetration without using
chemicals or viruses.
In brief, biolistic particle delivery, also known as particle bombardment, involves

projecting microscopic heavy-metal particles (often gold or tungsten) coated with
nucleic acids into recipient cells at high velocity using a ballistic device (i.e., “gene
gun”). Biolistic particle delivery can be used to transiently transfect dividing and
non‑dividing cells in culture as well as cells in vivo, and it is often used for genetic
vaccination and agriculture applications (Klein et al., 1992; Ye et al., 1990; Burkholder
et al., 1993). While this technique is reliable and fast, it requires costly equipment,
causes physical damage to the samples, and necessitates high cell numbers due to high
mortality.
Direct microinjection delivers nucleic acids into the cytoplasm or the nucleus one
cell at a time by means of a fine needle; therefore, this method is limited to ex vivo
applications such as the transfer of genes into oocytes to engineer transgenic animals or
the delivery of artificial chromosomes (Cappechi, 1980; Cappechi, 1989; Telenius et al.,
1999). Although direct microinjection is nearly 100% efficient, it demands considerable
technical skill, is extremely labor-intensive, and often causes cell death. As such, this
method is not appropriate for studies that require the transfection of large number of
cells.
Laser-mediated transfection, also known as phototransfection, laserfection, or

optoporation, uses a laser pulse to transiently permeabilize the cell membrane (Shirahata
et al., 2001; Schneckenburger et al., 2002). When the laser induces a pore in the membrane,
the osmotic difference between the medium and the cytosol facilitates the entry of
nucleic acids or other desired substances in the medium (ions, small molecules, proteins,
semiconductor nanocrystals, etc.) into the cell. Advantages of laser-mediated transfection
include high transfection efficiency and the ability to make pores at any location on the
cell. However, the method requires an expensive laser-microscope system and the cells to
be attached to a substrate.
In addition to the methods mentioned above, other physical delivery technologies use
hydrodynamic pressure, ultrasound, or magnetic field to drive naked nucleic acids or
nucleic acid-particle complexes into recipient cells.

Cationic Lipid-Mediated Transfection

Mechanism Specially designed cationic lipids, such as the Lipofectamine® Transfection Reagents,
facilitate DNA and siRNA delivery into cells (Chesnoy and Huang, 2000; Hirko et al.,
2003; Liu et al., 2003). The basic structure of cationic lipids consists of a positively
charged head group and one or two hydrocarbon chains. The charged head group
governs the interaction between the lipid and the phosphate backbone of the nucleic
acid, and facilitates DNA condensation. Often, cationic lipids are formulated with
a neutral co-lipid or helper lipid, followed by extrusion or microfluidization, which
results in a unilamellar liposomal structure with a positive surface charge when in
water.
The positive surface charge of the liposomes mediates the interaction of the nucleic acid
and the cell membrane, allowing for fusion of the liposome/nucleic acid transfection
complex with the negatively charged cell membrane. The transfection complex is
thought to enter the cell through endocytosis. Endocytosis is the process where a
localized region of the cellular membrane uptakes the DNA:liposome complex by
forming a membrane bound/intracellular vesicle. Once inside the cell, the complex
must escape the endosomal pathway, diffuse through the cytoplasm, and enter the
nucleus for gene expression. Cationic lipids are thought to facilitate transfection during
the early steps of the process by mediating DNA condensation and DNA/cellular
interactions.
The principle of delivery using cationic lipid reagents thus differs from prior attempts
to use neutral liposomes for transfections. With cationic lipid reagents, the DNA
solution is not deliberately encapsulated within the liposomes; rather, the negatively
charged DNA binds spontaneously to the positively charged liposomes, forming DNA-
cationic lipid reagent complexes.
Some of the problems associated with traditional transfection methods like calcium
phosphate co-precipitation, DEAE-dextran, polybrene, and electroporation include
low efficiency of DNA delivery, poor reproducibility, cell toxicity, and inconvenience.
In contrast, cationic lipid reagent-mediated transfection yields high and previously
unattainable transfection efficiencies in a wide variety of eukaryotic cells. It is simple to
perform, and ensures consistently reproducible results. Moreover, a number of cell lines
normally resistant to transfection by other methods transfect successfully with cationic
lipid reagents.
Cationic Lipid
Transfection Reagent
DNA
DNA-Cationic lipid Interaction of the complex with Expression following
complex formation the mammalian cell line successful transfection
Figure 5.7 Mechanism of cationic lipid-mediated delivery.
55
Cationic lipid transfection

reagents Cationic lipid-mediated delivery is a fast, simple, and reproducible means for easily
introducing DNA, RNA, siRNA, or oligonucleotides into eukaryotic cells. It allows
the highly efficient transfection of a broad range of cell types, including adherent,
suspension, and insect cells, as well as primary cultures. When selecting a transfection
reagent, you must consider the payload you wish to deliver (DNA, RNA, or protein)
and the type of cells you want to transfect, because the choice of the transfection
reagent strongly influences transfections results.
The table below lists the key features and applications of various cationic-lipid
transfection reagents available from Life Technologies™. For more information on each
transfection reagent and for optimized transfection protocols for a wide range of cell
lines, go to www.lifetechnologies.com/transfection.
Transfection Reagent Payload Key Features and Applications

Broad-Spectrum Reagents
• Highest efficiency and expression results for plasmid and RNAi cotransfections
• High transfection efficiency even at low doses
Lipofectamine® 3000 • Works effectively with a wide range of cell types, both adherent and suspension
• No need for wash steps before or after transfection
• Complexes can be added to cells growing in serum-containing media
• High efficiency and expression results for plasmid or RNAi transfections

• Works effectively with a wide range of cell types, both adherent and suspension
• For robust cells
• No need for wash steps before or after transfection
Lipofectamine® 2000 • Complexes can be added to cells growing in serum-containing media
• Ideal at >90% confluency at the time of transfection
• Recommended for delivery of Stealth RNAi® and Silencer® Select siRNAs,
dicer-generated siRNA pools and plasmids containing shRNA cassettes
• High-throughput
• Same performance as Lipofectamine® 2000, certified animal-origin free

Lipofectamine® 2000 CD
(“CD” = chemically defined)
• Highest transfection efficiency in Chinese Hamster Ovary (CHO) cells

• High transfection efficiency and significantly lower toxicity for a wide range of cell lines
• Very gentle to cells
• Wide range of cell lines, including primary and disease-related cells
Lipofectamine® LTX • High protein expression
with PLUS™ Reagent
• Delivery of shRNA and miR RNAi vectors
• Significantly improved transfection performance in a number of primary and
hard-to-transfect cell lines
• Optimized protocols for over 30 different cell lines are available, so much less time is
needed for evaluation and optimization
Lipofectamine® • First generation reagent for plasmid DNA transfections
Lipofectin® • First generation reagent for plasmid DNA transfections

Transfection Reagent Payload Key Features and Applications

RNA Reagents
• Recommended for transient delivery of Stealth RNAi® and Silencer® Select siRNAs,
dicer-generated siRNA pools, mirVana™ miRNA Mimics and Inhibitors, mRNA, and snRNA
• Requires lower RNAi concentrations leading to more effective gene knockdown with minimal
non-specific effects
Lipofectamine® • Minimal cytotoxicity across a 10-fold concentration range of transfection reagent
RNAiMAX
• Compatibility with a broad range of cell types
• High-throughput
• Highest knockdown with less RNAi
• in vitro transfection
• Highest transfection efficiency in neurons (> 2-fold) and a broad spectrum of primary cell types
Lipofectamine®
• Enhanced gene editing outcomes using mRNA CRISPRs
MessengerMAX
• Faster protein expression with no risk of genomic integration
• Efficiently delivers siRNA in vivo through systemic delivery

®
Invivofectamine 2.0 • Highly effective mRNA, protein, and functional knockdown
• Low toxicity profile and easy to use
• Transfection of antisense oligonucleotides

• High-throughput
Oligofectamine®
• Highly-specific, non-toxic
• Ideal for low confluency (30–50% confluent at the time of transfection)
Protein Production Reagents

• Transfection of high density 293 suspension cell culture for bioproduction
• Transfection enhancers boost performance and protein expression with yields 2- to 10-fold higher
®
than other transfection reagents used on high density 293 cell cultures
ExpiFectamine 293
• Robust and reproducible transfection results
• Scale transfections for culture volumes of less than 1 mL to greater than 10 liters, while
maintaining equivalent volumetric protein yields
• Used for rapid large-scale transient mammalian protein expression for bioproduction
™
FreeStyle MAX • High-yield production with milligrams of protein yield
• Optimized for transient transfection in CHO suspension cells and also works for HEK-293 cells
• Used for transient protein bioproduction in combination with the FreeStyle™ 293 Expression System
293fectin™
• Optimized for suspension FreeStyle™ 293-F cells
• Ideal for low confluency (<70% confluent at the time of transfection)

Optifect™
• For cell lines that are sensitive to transfection reagents
Cellfectin® • Optimal transfection of insect cells, including S2, Sf9, Sf21 and High Five™ cells
DMRIE-C Reagent • Transfection of suspension cells, including CHO, lymphoid and Jurkat cell lines
Symbol Explanation Symbol Explanation Symbol Explanation
Plasmid DNA for expression of Non-coding RNA for RNAi Co-delivery for cotransfection of
protein, shRNA, and miRNA inhibition of gene expression RNAi vectors and siRNAs
Plasmid DNA for expression of Oligonucleotides for antisense

mRNA for expression of protein
protein inhibition of gene expression
57
Virus-Mediated Gene Transfer

For cell types not amenable to lipid-mediated transfection, viral vectors are often
employed. Virus-mediated transfection, also known as transduction, offers a means
to reach hard-to-transfect cell types for protein overexpression or knockdown, and
it is the most commonly used method in clinical research (Glover et al., 2005; Pfeifer
and Verma, 2001). One of the main advantages of viral delivery is that the process can
be performed inside a living organism (in vivo) or in cell culture (in vitro) with gene
delivery efficiencies approaching 95–100%.
Key properties of viral

vectors Viral vectors are tailored to their specific applications, but must generally share a few
key properties.
• Safety: Although viral vectors are occasionally created from pathogenic viruses, they
are modified in such a way as to minimize the risk of handling them. This usually
involves the deletion of a part of the viral genome critical for viral replication, allowing
the virus to efficiently infect cells and deliver the viral payload, but preventing the
production of new virions in the absence of a helper virus that provides the missing
critical proteins. However, an ongoing safety concern with the use of viral vectors is
insertional mutagenesis, in which the ectopic chromosomal integration of viral DNA
either disrupts the expression of a tumor-suppressor gene or activates an oncogene,
leading to the malignant transformation of cells (Glover et al., 2005).
• Low toxicity: The viral vector should have a minimal effect on the physiology of the
cell it infects. This is especially important in studies requiring gene delivery in vivo,
because the organism will develop an immune response if the vector is seen as a
foreign invader (Nayak and Herzog, 2009).
• Stability: Some viruses are genetically unstable and can rapidly rearrange their
genomes. This is detrimental to predictability and reproducibility of the work
conducted using a viral vector. Therefore, unstable vectors are usually avoided.
• Cell type specificity: Most viral vectors are engineered to infect as wide a range
of cell types as possible. However, sometimes the opposite is preferred. The viral
receptor can be modified to target the virus to a specific kind of cell. Viruses
modified in this manner are said to be pseudotyped.
• Selection: Viral vectors should contain selectable markers, such as resistance to a
certain antibiotic, so that the cells that have taken up the viral vector can be isolated.
Common viral vectors Adenoviruses are DNA viruses with broad cell tropism that can transiently transduce
nearly any mammalian cell type. The adenovirus enters target cells by binding to the
Coxsackie/Adenovirus receptor (CAR) (Bergelson et al.,1997). After binding to the CAR,
the adenovirus is internalized via integrin-mediated endocytosis followed by active
transport to the nucleus, where its DNA is expressed episomally (Hirata and Russell,
2000). Although adenoviral vectors work well for transient delivery in many cell types,
for some difficult cell lines such as non‑dividing cells and for stable expression, lentiviral
vectors are preferred. The packaging capacity of adenoviruses is 7–8 kb.
Retroviruses are positive-strand RNA viruses that stably integrate their genomes into
host cell chromosomes. When pseudotyped with an envelope that has broad tropism,
such as vesicular stomatitis virus glycoprotein (VSV-G), these viruses can enter virtually
any mammalian cell type. However, most retroviruses depend upon the breakdown
of nuclear membrane during cell division to infect cells and are thus limited by the
requirement of replicating cells for transduction. Other disadvantages of retroviruses
include the possibility of insertional mutagenesis and the potential for the activation of
latent disease. Like adenoviruses, retroviruses can carry foreign genes of around 8 kb.

Lentiviruses are a subgroup of the retrovirus family; as such, they can integrate into
the host cell genome to allow stable, long-term expression (Anson, 2004). In contrast
to other retroviruses, lentiviruses are more versatile tools as they use an active nuclear
import pathway to transduce non-dividing, terminally differentiated cell populations
such as neuronal and hematopoietic cells.
Adeno-associated viruses are capable of transducing a broad range of dividing

and non-dividing cells types, but they require coinfection with a helper virus like
adenovirus or herpes virus to produce recombinant virions in packaging cells. This
causes difficulties in obtaining high quality viral stocks that are free of helper viruses.
Furthermore, adeno-associated viruses have only limited packaging capacity of up to
4.9 kb. On the other hand, adeno-associated viruses show low immunogenicity in most
cell types, and they have the ability to integrate into a specific region of the human
chromosome, thereby avoiding insertional mutagenesis.
Other viral vector systems that can be used for overexpression of proteins include
vectors based on baculovirus, vaccinia virus, and herpes simplex virus. While
baculoviruses normally infect insect cells, recombinant baculoviruses can serve as
gene‑transfer vehicles for transient expression of recombinant proteins in a wide range
of mammalian cell types. Furthermore, by including a dominant selectable marker in
the baculoviral vector, cell lines can be derived that stably express recombinant genes
(Condreay et al., 1999). Vectors based on vaccinia virus can be used for introducing
large DNA fragments into a wide range of mammalian cells. However, cells infected
with vaccinia virus die within one or two days, limiting this system to transient protein
production. Herpex simplex viruses are a class of double-stranded DNA viruses that
infect neurons.
DNA insert Maximum titer

Viral system Size Infection Expression Drawbacks
size (particles/mL)
36 kb Dividing and Elicits strong antiviral

Adenovirus 8 kb 1 × 1013 Transient
(dsDNA) non‑dividing cells immune response
7–11 kb Insertional mutagenesis

Retrovirus 8 kb 1 × 109 Dividing cells Stable
(ssRNA) potential
8 kb Dividing and Insertional mutagenesis

Lentivirus 9 kb 1 × 109 Stable
(ssRNA) non‑dividing cells potential
Stable; Requires helper virus for

Adeno-associated 8.5 kb Dividing and
5 kb 1 × 1011 site‑specific replication; difficult to
virus (ssDNA) non‑dividing cells
integration produce pure viral stocks
80–180 kb no known Dividing and Transient or Limited mammalian host

Baculovirus 2 × 108
(dsDNA) upper limit non‑dividing cells stable range
190 kb Potential cytopathic

Vaccinia virus 25 kb 3 × 109 Dividing cells Transient
(dsDNA) effects
Herpex simplex 150 kb Dividing and No gene expression

30–40 kb 1 × 109 Transient
virus (dsDNA) non‑dividing cells during latent infection
59
Neon® Transfection System

The Neon® Transfection System, a second-generation benchtop electroporation device
offered by Life Technologies™, uses an electronic pipette tip as an electroporation
chamber to efficiently transfect mammalian cells including primary and immortalized
hematopoietic cells, stem cells, and primary cells. The design of the electroporation
chamber distributes the current equally among the cells and maintains a stable pH
throughout the chamber, resulting in less ion formation and negligible heat generation
for increased cell viability and transfection efficiency compared to traditional cuvette-
based electroporation systems.
The Neon® Transfection System efficiently delivers nucleic acids, proteins, and siRNA
into all mammalian cell types, including primary and stem cells, with a high cell
survival rate. The transfection is performed using as few as 1 × 104 or as many as 5 × 106
cells per reaction using a sample volume of 10 μL or 100 μL in a variety of cell culture
formats (60‑mm, 6-well, 48-well, and 24-well).
Because the Neon® Transfection System uses a single transfection kit (Neon® Kit) that
is compatible with various mammalian cell types including primary and stem cells,
the need to determine an optimal buffer for each cell type is avoided. Furthermore,
the Neon® device is pre-programmed with a 24-well optimization protocol to optimize
conditions for nucleic acid/siRNA and cell type, and allows the programming and
storage of up to 50 cell-specific protocols in the Neon® device database. Optimized
protocols can also be conveniently downloaded from www.lifetechnologies.com/neon
to maximize transfection efficiencies for many commonly used cell types.
Cell types successfully transfected with the Neon® Transfection System*
Blood/Immune Cells
Human SKW6.4 Human Macrophage

Human BC-1 Cells Human Jiyoye Cells Human Raji Cells Human HL-60 Cells
Cells Cells
Human RPMI8226 Human NAMALWA

Human BJAB Cells Human K-562 Cells Human Ramos Cells Human KG-1 Cells
Cells Cells
Human Dendritic Human CCRF-CEM

Human IM-9 Cells Human LCL Cells Human RS4-11 Cells Human PBMC Cells
Cells Cells
Human SCID.adh Mouse RAW 264.7

Human U-937 Cells Human Jurkat Cells Mouse MPC-11 Cells Mouse Ramos Cells
Cells Cells
Mouse RBL-2H3 Mouse BW5147

Mouse M1 Cells Mouse EL4 Cells Mouse P815 Cells
Cells (T200-A)5.2 Cells
Connective Tissue
Human HT-1080
Human HOS Cells Human MH7A Cells Human BJ Cells Human U-2 OS Cells Human IMR-90 Cells
Cells
Human Neonatal Mouse Embryonic

Mouse NIH-3T3
Human Saos-2 Cells Dermal Fibroblast Human WI-38 Cells Fibroblast Cells Mouse PA317 Cells
Cells
Cells Gibco® (MEF)
Horse Embryonic Dermal

Mouse L-929 Cells Mouse 3T3-L1 Cells Monkey COS-7 Cells Monkey Vero Cells
Fibroblast Cells (NBL-6)
* For optimized Neon® transfection protocols using the cell types listed here, go to www.lifetechnologies.com/neon.

Cell types successfully transfected with the Neon® Transfection System, continued*
Epithelial Cells
Human ChangX-31 Human HEK 293 Human ARPE-19 Human COLO 201 Human HCT 116
Human T24 Cells
Cells Cells Cells Cells Cells
Human 253J Cells Human HT-29 Cells Human HCT15 Cells Human RKO Cells Human SW480 Cells Human WiDr Cells
Human 293A Cells Human J82 Cells Human RT4 Cells Human Hep G2 Cells Human Hep3B Cells Human BT-20 Cells
Human Mammary Human SK-HEP-1 Human SNU-387 Human HCC1937 Human Hs-578T
Human MCF7 Cells
Epithelial Cells Gibco® Cells Cells Cells Cells
Human MDA- Human SK-BR-3 Human SK-OV-3 Human MCF-ADR

Human T-47D Cells Human DU 145 Cells
MB-231 Cells Cells Cells Cells
Human PANC-1 Human BxPC-3 Human NCI-H23

Human LNCaP Cells Human A549 Cells Human PC-3 Cells
Cells Cells Cells
Human TSU-Pr1 Human BEAS-2B Human NCI-H69

Human HN3 Cells Human G-361 Cells Human ARO Cells
Cells Cells Cells
Human HeLa Cells

Human FRO Cells Human Calu-3 Cells Human MEWO Cells Human NPA Cells Human A-431 Cells
(ATCC)
Human C-33 A Cells Mouse P19 Cells Rat GH3 Cells Rat NRK Cells Rat PC-12 Cells Rat H-4-II-E Cells
Chinese Hamster Chinese Hamster Hamster BHK-21

Canine MDCK Cells
CHO-K1 Cells CHO DG44 Cells Cells
Endothelial Cells
Human Endothelial Mouse b-END.3

Human HUVEC Cells
Cells Gibco® Cells
Muscle Cells
Human Aortic Smooth Rat Cardiomyocyte

Mouse C2C12 Cells Rat L6 Cells
Muscle Cells Gibco® Cells
Neural/Glial Cells
Human U-87 MG Human SK-N-MC Human SH-SY5Y

Human T98G Cells Mouse GT1-1 Cells Mouse GT1-7 Cells
Cells Cells Cells
Rat Cortical Rat Primary

Rat Primary Cortical Rat Glial Precursor
Mouse Glial Cells Astrocyte Cells Rat Astrocyte Cells Hippocampal Neuron
Neuron Cells Gibco® Cells Gibco®
Gibco® Cells Gibco®
Rat HiB5 Cells Rat C6 Glial Cells Rat SCN2.2 Cells Rat F-11 Cells
Secretory Cells
Human SW-13 Cells Human SV40 MES 13 Cells
Stem Cells
Human Adipose-
Human Mesenchymal Human BGO1V Human H9 Embryonic Human Neural Stem Mouse Embryonic
derived Stem Cells
Stem Cells (hMSC) Embryonic Stem Cells Stem Cells Cells Gibco® Stem Cells
(ADSC)
Rat Neural Stem

Cells Gibco®
* For optimized Neon® transfection protocols using the cell types listed here, go to www.lifetechnologies.com/neon.
61
Selection of Stable Transfectants

Successful stable transfection requires both effective DNA delivery and a way to select
cells that have acquired the DNA. Approximately one in 104 transfected cells will stably
integrate DNA, although the efficiency varies with cell type and whether linear or
circular DNA is used. Integration is most efficient when linear DNA is used.
One of the most reliable ways to select cells that stably express transfected DNA is to
include a selectable marker on the DNA construct used for transfection or on a separate
vector that is co-transfected into the cell, and then apply the appropriate selective
pressure to the cells after a short recovery period. When the selectable marker is
expressed from the co-transfected vector, the molar ratio of the vector carrying the gene
of interest to the vector carrying the selectable marker should be in the range of 5:1 to
10:1 to ensure that any cell that contains the selectable marker also contains the gene of
interest.
Frequently used selectable markers are genes that confer resistance to various selection
drugs or genes that compensate for an essential gene that is defective in the cell line
to be transfected. When cultured in selective medium, cells that were not transfected
or were transiently transfected will die, and those that express the antibiotic resistance
gene at sufficient levels or those that can compensate for the defect in the essential gene
will survive.
Selection antibiotics for

eukaryotic cells Life Technologies™ offers high-quality selection reagents to complement its wide
variety of selectable eukaryotic expression vectors. Geneticin® (G418 sulfate), Zeocin™,
hygromycin B, puromycin, and blasticidin antibiotics are the most commonly used
selection antibiotics for stable cell transfection. These antibiotics provide unique
solutions for your research needs, such as dual selection and rapid, stable cell line
establishment.
Geneticin® Selection Antibiotic

Geneticin® reagent, also known as G418 sulfate, is commonly used for the selection of
mammalian, plant, or yeast cells. The higher purity of Geneticin® reagent available from
Life Technologies™ means that 15–30% lower concentrations are required compared
to other G418 products; therefore, surviving clonal colonies may arise faster, and cells
appear healthier.
Zeocin™ Selection Antibiotic

Zeocin™ reagent is effective in mammalian cell lines, yeast, insect cells, and bacteria.
Resistance to Zeocin™ reagent is conferred by the She ble gene, which prevents the
binding of Zeocin™ reagent and cleavage of cellular DNA in cells expressing the protein.
The concentration required for selection ranges from 50 to 2,000 μg/mL (typically
300 μg/mL), depending on the cell type.
Hygromycin B Selection Antibiotic

Hygromycin B is an aminoglycosidic antibiotic that inhibits protein synthesis by
disrupting translocation and promoting mistranslation of the 80S ribosome. Because its
mode of action is different from Geneticin® or Zeocin™ reagents, hygromycin B can be
used in dual-selection experiments. Resistance to hygromycin B is conferred by the E. coli
hygromycin resistance gene (hyg or hph). The concentration for selection ranges from
100 to 1,000 μg/mL (typically 200 μg/mL), and should be optimized for each cell line.

Puromycin Dihydrochloride Selection Antibiotic

Puromycin, a translation inhibitor in both prokaryotic and eukaryotic cells, is an
aminonucleoside antibiotic from Streptomyces alboniger. Resistance is conferred by the
puromycin N-acetyltransferase gene (pac) from Streptomyces. Puromycin has a fast mode
action, causing rapid cell death even at low antibiotic concentrations, allowing the
generation of puromycin-resistant stable cell lines in less than one week. Adherent
mammalian cells are sensitive to concentrations of 2–5 μg/mL, while cells in suspension
are sensitive to concentrations as low as 0.5–2 μg/mL.
Blasticidin S HCl Selection Antibiotic

Blasticidin, a potent translational inhibitor in both prokaryotic and eukaryotic cells, is
a nucleoside antibiotic from Streptomyces griseochromogenes. Resistance is conferred by
the bsd gene product from Aspergillus terreus. E. coli strains are generally sensitive to
concentrations of 50 μg/mL, while mammalian cells are sensitive to concentrations
as low as 2–10 μg/mL. Cell death occurs rapidly in cells sensitive to blasticidin, and
blasticidin-resistant, stable mammalian cell lines can be generated in less than one week
at low antibiotic concentrations.
Reporter Gene Assays

Reporter genes are genes whose products can be readily assayed subsequent to
transfection, and can be used as markers for screening successfully transfected cells,
for studying regulation of gene expression, or serve as controls for standardizing
transfection efficiencies.
The ideal reporter gene should be absent from the cells used in the study or easily
distinguishable from the native form of the gene, assayed conveniently, and have a
broad linear detection range. It is also important that the presence of the reporter gene
does not affect the normal physiology and general health of the transfected cells.
Reporter genes can be expressed constitutively or inducably with an external

intervention such as the introduction of IPTG in the β-galactosidase system. Generally,
reporter gene assays are performed 1–3 days after transfection; however the optimal
time for the assay should be determined empirically.
Transfection assays In contrast to selectable markers, which protect an organism from a selective agent
that would normally kill it or prevent its growth, reporter genes used for screening
transfectants make the cells containing the reporter gene visually identifiable. Reporter
genes used in this way are normally expressed under their own promoter independent
from that of the introduced gene of interest, allowing the screening of successfully
transfected cells even when the gene of interest is only expressed under certain specific
conditions or in tissues that are difficult to access.
Reporter genes can also serve as controls for transfection. For example, transfection
efficiencies between different experiments can be normalized by comparing the
expression levels of a reporter gene used in all of the experiments.
63
Gene regulation assays Reporter gene assays are invaluable for studying regulation of gene expression, both
by cis-acting factors (gene regulatory elements) and trans-acting factors (transcription
factors or exogenous regulators). Furthermore, reporter gene systems enable the use
of pathway-specific, tissue-specific, or developmentally regulated gene promoters as
biomarkers for specific events processes.
In these assays, the detectable reporter gene acts as a surrogate for the coding region of
the gene under study. The reporter gene construct contains one or more gene regulatory
elements to be analyzed, the sequence for the reporter gene, and the sequences required
for the transcription of functional mRNA. Upon introduction of the reporter construct
into cells, expression levels of the reporter gene are monitored through a direct assay of
the reporter proteins enzymatic activity.
Common reporter genes Commonly used reporter genes that induce visually identifiable characteristics usually
involve fluorescent and luminescent proteins.
Green fluorescent protein (GFP) causes cells that express it to glow green under UV
light. A specialized microscope is required to see individual cells. Yellow and red
versions are also available, allowing the investigation of multiple genes at once. It is
commonly used to measure gene expression.
Luciferase as a laboratory reagent often refers to P. pyralis luciferase, although

recombinant luciferases from several other species of fireflies are also commercially
available. The luciferase enzyme catalyzes a reaction with its substrate (usually
luciferin) to produce yellow-green or blue light, depending on the luciferase gene.
Since light excitation is not needed for luciferase bioluminescence, there is minimal
autofluorescence and thus virtually background-free fluorescence.
GUS assay (using β-glucuronidase) is an excellent method for detecting a single cell
by staining it blue without using any complicated equipment. The drawback is that the
cells are killed in the process. It is particularly common in plant science.
Blue-white screen is used in both bacteria and eukaryotic cells. The bacterial lacZ gene
encodes a β-galactosidase enzyme. When media containing certain galactosides (e.g.,
X-gal) is added, cells expressing the gene convert the X-gal to a blue product and can be
seen with the naked eye.

RNAi and Non-coding RNA Research

RNA interference (RNAi) is a very powerful tool for studying the basic biology of cells,
allowing the knockdown of gene expression to study protein function in a wide range
of cell types. Once viewed as a technique used only by select laboratories, RNAi is now
considered essential for studying gene function. It has become a prominent tool for
protein knockdown studies, phenotype analysis, function recovery, pathway analysis, in
vivo knockdown, and drug target discovery.
Glossary of common
RNAi terms RNAi
Ribonucleic acid interference (first used by A. Fire and C. Mello et al., 1998).
siRNA
Short interfering RNA. siRNAs are 21–25 bp dsRNAs with dinucleotide 3’ overhangs
and are processed from longer dsRNA by Dicer in the RNA interference pathway.
Introduction of synthetic siRNAs can induce RNAi in mammalian cells. siRNAs can
also originate from endogenous precursors.
shRNA
Short hairpin RNA; also short interfering hairpin. shRNAs are used in vector-based
approaches for supplying siRNA to cells for stable gene silencing. A strong Pol III-type
promoter is used to drive transcription of a target sequence designed to form hairpins
and loops of variable length, which are processed by cellular siRNA machinery. Once
in the cell, the shRNA can decrease the expression of a gene with complementary
sequences by RNAi.
miR RNAi
Vectors that express microRNAs for RNAi. miRNAs are 19–23 nt single-stranded
RNAs, originating from single-stranded precursor transcripts that are characterized
by imperfectly base-paired hairpins. miRNAs function in a silencing complex that is
similar, if not identical, to RISC (see below).
Chemically modified siRNA

siRNA molecules which have chemical modifications.
RISC
RNA-induced silencing complex (RISC). A nuclease complex composed of proteins
and siRNA that targets and cleaves endogenous mRNAs complementary to the siRNA
within the RISC complex.
Off-target effects
Effects that occur when one or a few genes not specifically targeted show loss of gene
function following the introduction of an siRNA or d-siRNA pool. The effect may
be mediated by the sense strand of an siRNA, which may initiate a loss-of-function
response from an unrelated gene. Off-target effects can also occur as a secondary effect
of the antisense strand of a specific siRNA, if it has sufficient homology to knock down
the expression of a non-target gene.
65
How RNAi works Two types of small RNA molecules function in RNAi. The first are synthetic, short
interfering RNA (siRNA) molecules that target mRNA cleavage, effectively knocking
down the expression of a gene of interest. MicroRNA (miRNA) molecules, on the other
hand, are naturally occurring single-stranded RNAs 19–22 nucleotides long, which
regulate gene expression by binding to the 3’ untranslated regions (UTRs) of target
mRNAs and inhibiting their translation (Ambros, 2004). For more information on RNAi,
go to www.lifetechnologies.com/rnai.
siRNA analysis There are several ways to induce RNAi: synthetic molecules, RNAi vectors, and in
vitro dicing (Figure 5.8, below). In mammalian cells, short pieces of dsRNA—short
interfering RNA— initiate the specific degradation of a targeted cellular mRNA. In
this process, the antisense strand of siRNA becomes part of a multiprotein complex,
or RNA-induced silencing complex (RISC), which then identifies the corresponding
mRNA and cleaves it at a specific site. Next, this cleaved message is targeted for
degradation, which ultimately results in the loss of protein expression. For more
information on siRNA analysis, go to www.lifetechnologies.com/sirna.
Synthetic molecules for RNAi Vector-based expressed RNAi In vitro synthesized RNAi
Silencer® Select Stealth™ RNAi™

shRNA expression miRNA expression Dicer processing
siRNA siRNA
of long dsRNA
Dicer
siRNA in mammalian cell

(pool of 21–23 nt siRNAs)
RISC loading
siRNA unwinding
Target recognition
Target mRNA
Target cleavage
Cellular message is destroyed
Figure 5.8 Methods of RNAi knockdown in mammalian cells.

miRNA analysis Both RNA polymerase II and III transcribe miRNA-containing genes, generating long
primary transcripts (pri-miRNAs) that are processed by the RNase III–type enzyme
Drosha, yielding hairpin structures 70 to 90 bp in length (pre-miRNAs). Pre‑miRNA
hairpins are exported to the cytoplasm, where they are further processed by the RNase III
protein Dicer into short double-stranded miRNA duplexes 19 to 22 nucleotides long. The
miRNA duplex is recognized by the RNA-induced silencing complex (RISC), a multiple-
protein nuclease complex, and one of the two strands, the guide strand, assists this
protein complex in recognizing its cognate mRNA transcript. The RISC-miRNA complex
often interacts with the 3’ UTR of target mRNAs at regions exhibiting imperfect sequence
homology, inhibiting protein synthesis by a mechanism that has yet to be fully elucidated
(Figure 5.9, below).
Plant miRNAs can bind to sequences on target mRNAs by exact or near-exact

complementary base pairing and thereby direct cleavage and destruction of the
mRNA (Rhoades et al., 2002; Chen, 2005). Similar to the mechanism employed in RNA
interference (RNAi), the cleavage of a single phosphodiester bond on the target mRNA
occurs between bases 10 and 11 (Elbashir et al., 2001). In contrast, nearly all animal
miRNAs studied so far do not exhibit perfect complementarity to their mRNA targets,
and seem to inhibit protein synthesis while retaining the stability of the mRNA target
(Ambros, 2004). It has been suggested that transcripts may be regulated by multiple
miRNAs, and an individual miRNA may target numerous transcripts. Research
suggests that as many as one-third of human genes may be regulated by miRNAs
(Lim et al., 2003). Although hundreds of miRNAs have been discovered in a variety
of organisms, little is known about their cellular function. Several unique physical
attributes of miRNAs, including their small size, lack of polyadenylated tails, and
tendency to bind their mRNA targets with imperfect sequence homology, have made
them elusive and challenging to study. For more information on miRNA analysis, go to
www.lifetechnologies.com/mirna.
Nuclear
pore
miRNA gene
Xpo-5
Dicer
Pol II and Pol III
mRNA transcription Drosha
i
miRNA
Precursor miRNA Unwinding

m7Gppp AAAAAA (pre-miRNA)
Primary miRNA
(pri-miRNA) RISC RISC
Nucleus 3´ UTR
miRNA has 100% homology to mRNA,
Cytoplasm which results in target mRNA cleavage
miRNA binds 3´ UTR with imperfect
homology, inhibiting translation
Figure 5.9 Biogenesis and function of miRNA. MicroRNA transcripts, generated by RNA polymerases II and III, are
processed by the RNase III enzymes Drosha (nuclear) and Dicer (cytoplasmic), yielding 19–22 nucleotide miRNA
duplexes. One of the two strands of the duplex is incorporated into the RISC complex,which regulates protein expression.
67
Choosing an RNAi
approach The process of RNAi (RNA interference) can be moderated by either siRNA or miRNA.
Both are processed inside the cell by the enzyme called Dicer and incorporated into
a complex called RISC (RNA-induced silencing complex). However, there are subtle
differences between the two.
siRNA is an exogenous double-stranded RNA that can either be chemically synthesized

and then directly transfected into cells, or generated inside the cell by introducing
vectors that express short-hairpin RNA (shRNA), which are the precursors of siRNAs.
miRNA, on the other hand, is single stranded and comes from endogenous non-coding
RNA found within the introns of larger RNA molecules. However, the processing of
shRNA into functional siRNA involves the same cellular RNAi machinery that naturally
processes genome-encoded miRNAs, which are responsible for cellular regulation of
gene expression by modulating mRNA stability, translation, and chromatin structures
(Hutvagner and Zamore, 2002).
Another difference between siRNA and miRNA is that siRNA typically binds perfectly
and specifically to its mRNA target in animals, while miRNA can inhibit translation
of many different mRNA sequences because its pairing is imperfect. In plants, miRNA
tends to have a more perfectly complimentary sequence, which induces mRNA
cleavage as opposed to just repression of translation.
Both siRNA and miRNA can play a role in epigenetics through a process called RNA-
induced transcriptional silencing (RITS). Likewise, both are important targets for
therapeutic use because of the roles they play in the controlling gene expression.
siRNA miRNA
Occurs naturally in plants and lower animals. Whether Occurs naturally in plants and animals.
Occurrence or not they occur naturally in mammals is an unsettled
question.
Configuration Double stranded Single stranded
Length 21–22 nt 19–25 nt
Complementarity to 100% perfect match; therefore, siRNAs knock down Not exact; therefore, a single miRNA may target up to
target mRNA specific genes, with minor off-target exceptions. hundreds of mRNAs.
Regulate the same genes that express them. Expressed by genes whose purpose is to make miRNAs,
Biogenesis but they regulate genes (mRNAs) other than the ones
that expressed them.
Action Cleave mRNA Inhibit translation of mRNA
Act as gene silencing guardians in plants and animals Regulators (inhibitors) of genes (mRNAs)
Function
that do not have antibody-or cell-mediated immunity.
siRNAs are valuable laboratory tools used in nearly Possible therapeutic uses either as drug targets or as
every molecular biology laboratory to knock down drug agents themselves. Expression levels of miRNAs
Uses
genes. Several siRNAs are in clinical trials as possible can be used as potential diagnostic and biomarker tools.
therapeutic agents.
* Table adapted from Mack, 2007.

6. Transfection Methods
This section provides useful information and general guidelines for the transfection of
cells with plasmid DNA, oligonucleotides, and RNA, preparation of cultures for in vitro
and in vivo transfection, and selection of transfected cells.
Note that while the basics of transfection experiments share certain similarities,
conditions vary widely depending upon the cell type used for transfection. Therefore,
we recommend that you familiarize yourself with the cell line of interest and the
appropriate transfection method, and closely follow the instructions provided with each
product you are using in your experiments.
Factors Influencing Transfection Efficiency

Successful transfection is influenced by many factors—the choice of the transfection
method, health and viability of the cell line, number of passages, degree of confluency,
quality and quantity of the nucleic acid used, and the presence or absence of serum in
the medium can all play a part in the outcome of your transfection experiment. While
it is possible to optimize specific transfection conditions to achieve high transfection
efficiencies, it is important to note that some cell death is inevitable regardless of the
transfection method used.
Cell type The choice of which cell type to use for a transfection experiment may seem obvious,
but it is a critical factor that is often overlooked. Since each cell type is likely to respond
differently to a given transfection reagent or method, choosing the appropriate cell type
and proper experimental design are necessary to maximize results.
While established continuous cell lines are easier to work with in the laboratory, they
may not be the best choice for modeling in vivo processes because of the multiple
genetic changes that they have undergone. However, if the purpose of the transfection
experiment is high-level production of recombinant proteins, it is not important
that the cell line represents the in vivo situation as long as the cell line can express
sufficient quantities of recombinant proteins with proper folding and post-translational
modifications. For example, transient transfection of suspension-adapted Expi293F™
cells grown in Expi293™ Expression Medium enables researchers to produce, starting
from the vector of interest, greater than 1 g/L of correctly folded and glycosylated
recombinant proteins.
Primary cultures, on the other hand, are often used because they more closely mimic
natural tissues. However, they typically have a limited growth potential and life
span, and are more difficult to maintain in culture. When using primary cultures, it
is important to maintain a largely homogeneous population of cells (for example,
neuronal cultures should be enriched for neurons and suppressed with regard to glial
cells) and use the cells as soon as practical.
In addition, biological properties of the cell type must be taken into consideration when
designing transfection experiments. For example, some promoters function differently
in different cell types and some cell types are not well suited to particular transfection
technologies.

Part 6. Transfection Methods
Transfection efficiency in cancer cell line panel

100
Lipofectamine® 3000
% Transfection efficiency
80 Lipofectamine® 2000
60
40
20
NCI-H23
L6
Caki-1
DU-145
BT-549
NCI-H460
4T1
SK-MEL-28
Hs-578T
MDA-MB-468
PANC-1
Saos-2
RD
SK-OV-3
SW480
SW620
HCC1937
Cell line
Figure 6.1 Cell line-dependent differences in transfection efficiency. Lipofectamine® 2000 reagent and
Lipofectamine® 3000 reagent were used to transfect 17 cell lines with a GFP-expressing plasmid in a 24-well
plate format, using 0.5 µg plasmid/well and the recommended protocols for each reagent. GFP expression
was analyzed 48 hours posttransfection. Each condition was tested in triplicate, and the data points show
the mean transfection efficiency plus standard deviation.
Cell health and viability The viability and general health of cells prior to transfection is known to be an
important source of variability from one transfection to another. In general, cells should
be at least 90% viable prior to transfection and have had sufficient time to recover
from passaging. We strongly recommend subculturing cells at least 24 hours before
transfection to ensure that they recover from the subculture procedure and are in
optimum physiological condition for transfection.
Cell cultures with immortalized cell lines evolve over months and years in the
laboratory, resulting in changes in cell behavior with regard to transfection. Excessive
passaging is likely to detrimentally affect transfection efficiency as well as total
transgene expression level from the cell population as a whole. In general, we
recommend using cells that have undergone less than 30 passages after thawing of a
stock culture. Thawing a fresh vial of frozen cells and establishing low-passage cultures
for transfection experiments allow the recovery of transfection activity. For optimal
reproducibility, aliquots of cells of a low passage number can be stored frozen and
thawed as needed. Allow 3 or 4 passages after thawing a new vial of cells.
Since contamination can drastically alter transfection results, cell cultures and media
should be routinely tested for biological contamination (see Biological Contamination,
page 14), and contaminated cultures and media should never be used for transfection.
If cells have been contaminated or their health is compromised in any way, they should
be discarded and the culture re-seeded from uncontaminated frozen stocks.

Confluency For optimal transfection results, follow a routine subculturing procedure and passage
cultures once or twice a week at a dilution that allows them to become nearly confluent
before the next passage. Do not allow the cells to remain confluent for more than
24 hours.
The optimal cell density for transfection varies for different cell types, applications,
and transfection technology, and should be determined for every new cell line to be
transfected. Maintaining a standard seeding protocol from experiment to experiment
ensures that optimal confluency at the time of transfection is reliably achieved. With
cationic lipid-mediated transfection, generally 70–90% confluency for adherent cells or
5 × 105 to 2 × 106 cells/mL for suspension cells at the time of transfection provides good
results.
Make sure that the cells are not confluent or in stationary phase at the time of
transfection, because actively dividing cells take up foreign nucleic acid better than
quiescent cells. Too high of a cell density can cause contact inhibition, resulting in poor
uptake of nucleic acids and/or decreased expression of the transfected gene. However,
too few cells in culture may result in poor growth without cell-to-cell contact. In such
cases, increasing the number of cells in culture improves the transfection efficiency.
Similarly, actively dividing cell lines are more efficiently transduced with viral vectors.
When transducing a non-dividing cell type with viral constructs, the MOI (i.e.,
multiplicity of infection) may need to be increased to achieve optimal transduction
efficiency and increased expression levels for your recombinant protein.
Media Different cells or cell types have very specific medium, serum, and supplement
requirements, and choosing the most suitable medium for the cell type and transfection
method plays a very important role in transfection experiments. Information for
selecting the appropriate medium for a given cell type and transfection method is
usually available in published literature, and may also be obtained from the source of
the cells or cell banks. If there is no information available on the appropriate medium
for your cell type, you must determine it empirically.
It is important to use fresh medium, especially if any of the components are unstable,
because medium that is missing key components and necessary supplements may harm
cell growth.
For cell culture media information, see Media recommendations for common cell
lines, page 28, or refer to our website (www.lifetechnologies.com). Some cell lines
and primary cells may need special coating materials (e.g. poly-lysine, collagen,
fibronectin etc.) to attach to the culture plates and get the optimal transfection results.
Serum In general, the presence of serum in culture medium enhances transfection with DNA.
However, when performing cationic lipid-mediated transfection, it is important to form
DNA‑lipid complexes in the absence of serum because some serum proteins interfere
with complex formation. Note that the optimal amounts of cationic lipid reagent and
DNA may change in the presence of serum; thus, transfection conditions should be
optimized when using serum-containing transfection medium.
When transfecting cells with RNA, we recommend performing the transfection

procedure in the absence of serum to avoid possible contamination with RNases. Most
cells remain healthy for several hours in a serum-free medium.
71
The quality of serum can significantly affect cell growth and transfection result.
Therefore, it is important to control for variability among different brands or even
different lots of serum to obtain best results. After testing the serum on your cells, keep
using the same serum to avoid variation in your result. All Life Technologies™ and
Gibco® products, including sera, are tested for contamination and guaranteed for their
quality, safety, consistency, and regulatory compliance.
Antibiotics In general, antibiotics can be present in the medium for transient transfection. However,
because cationic lipid reagents increase cell permeability, they may also increase the
amount of antibiotics delivered into the cells, resulting in cytotoxicity and lower
transfection efficiency. Therefore, we do not recommend adding antibiotics to the
transfection medium. Avoiding antibiotics when plating cells for transfection also
reduces the need for rinsing the cells before transfection.
For stable transfections, penicillin and streptomycin should not be used in selective
medium, because these antibiotics are competitive inhibitors of the Geneticin® selective
antibiotic. When creating stable cell lines, allow 48 to 72 hours after the transfection
procedure for cells to express the resistance gene before adding the selective antibiotic.
If using serum-free medium, use lower amounts of antibiotics than you would in
serum-containing medium to maintain the health of the cells.
Type of molecule
transfected Plasmid DNA is the most commonly used vector for transfection. The topology (linear
or supercoiled) and the size of the plasmid DNA vector influence the efficiency of
transfection. Transient transfection is most efficient with supercoiled plasmid DNA.
In stable transfection, linear DNA results in lower DNA uptake by the cells relative to
supercoiled DNA, but yields optimal integration of DNA into the host genome.
Although other macromolecules such as oligonucleotides, RNA, siRNA, and proteins

can also be transfected into cells, conditions that work for plasmid DNA need to be
optimized when using other macromolecules.
Transfection method There are a number of strategies for introducing nucleic acids into cells that use various
biological, chemical, and physical methods. However, not all of these methods can be
applied to all types of cells and experimental applications, and there is a wide variation
with respect to transfection efficiency, cell toxicity, effects on normal physiology, level
of gene expression etc. The ideal approach should be selected depending your cell
type and experimental needs, and should have high transfection efficiency, low cell
toxicity, minimal effects on normal physiology, and be easy to use and reproducible.
For an overview and comparison of various transfection methods, see Gene Delivery
Technologies, page 46.

Selecting a Transfection Method (non-viral)

When selecting a transfection method, consider the payload you wish to deliver (DNA,
RNA, or protein) and the type of cells you want to transfect. Use the tables below
to choose between the various cationic-lipid transfection reagents and the Neon®
Transfection System available from Life Technologies™. For more information on each
transfection method, as well as optimized protocols for the transfection of wide range of
cell lines, go to www.lifetechnologies.com/transfection.
Continuous cell lines Continuous cell lines are capable of unlimited proliferative potential, and are generally
easier to work with than primary or finite cell cultures. However, because these cells
have undergone genetic transformation to become immortalized, their behavior in
culture may not necessarily reflect the in vivo situation.
Adherent cells
Transfection method Payload Suspension cells
easy-to-transfect hard-to-transfect
•••• •••• ••
reagent
••• •• •
reagent
Lipofectamine® RNAiMAX
•••• •••• ••
reagent
Lipofectamine® LTX with

•••• ••• •
PLUS™ reagent
Invivofectamine® 2.0
N/A N/A in vivo
reagent
Neon® electroporation •••• •••• ••••
Symbol Explanation Symbol Explanation Symbol Explanation Symbol Explanation

DNA for expression of Non-coding RNA for Co-delivery for
mRNA for expression
protein, shRNA, and RNAi inhibition of cotransfection of RNAi
of protein
miRNA gene expression vectors and siRNAs
73
Primary cells and finite

cultures Primary cells are isolated directly from the tissue and proliferated under appropriate
conditions. As such, they are morphologically and physiologically more similar to an
in vivo state. However, they are usually more difficult to culture and transfect than
continuous cell lines.
After the first subculture, the primary culture becomes known as a cell line. Cell lines
derived from primary cultures have a limited life span (i.e., they are finite), and as they
are passaged, cells with the highest growth capacity predominate, resulting in a degree
of genotypic and phenotypic uniformity in the population. Therefore, their phenotype
is intermediate between primary cells and continuous cultures. The use of such cells is
sometimes easier than the use of primary cells, especially for the generation of stably
transfected clones.
Transfection method Payload Neurons Stem cells Blood cells Others
••• ••• •• •••
reagent
••• •• • ••
reagent
Lipofectamine® RNAiMAX
••• ••• •• •••
reagent
Lipofectamine® LTX with

•• •• • •••
PLUS™ reagent
Invivofectamine® 2.0
N/A N/A in vivo
reagent
Neon® electroporation ••• ••• ••• •••

mRNA for expression
of protein

Selecting a Viral DNA Delivery System

There are many options in selecting a viral delivery system matched to your specific
needs. Life Technologies™ offers a variety of viral vector systems for delivering nucleic
acids into mammalian and insect cells for protein expression and RNAi studies.
Expression in
mammalian cells ViraPower™ Expression Systems from Life Technologies™ use replication-incompetent
viral particles to ensure safe and highly-efficient delivery of expression constructs for
high-level constitutive or inducible expression in any mammalian cell type. A number
of vectors available for use with the ViraPower™ systems offer various options for
cloning method (TOPO® or Gateway® cloning, or GeneArt® genetic assembly) and
promoter choice (constitutive or inducible), allowing the optimization of the experiment
for each cell line or animal model.
• ViraPower™ Lentiviral Expression System allows stable protein expression in

dividing and non-dividing cells (e.g., stem cells, primary neuronal cells), and are
ideal for analysis of long-term gene expression and functional analysis studies.
• ViraPower™ HiPerform™ Lentiviral Expression System improves on the existing
lentiviral systems by including the woodchuck posttranscriptional regulatory
element (WPRE) and the central polypurine tract(cPPT) sequence from the HIV-1
integrase gene in the viral vectors for increased expression and increased lentiviral
integration into the host genome, respectively. The ViraPower™ HiPerform™ kits
have two versions: kits for high accuracy titer, allowing for precise control of
copy number per cell, or kits for fast titering, which are ideal for high throughput
screening studies.
• ViraPower™ Lentiviral T-REx™ System combines the ViraPower™ HiPerform™
Lentiviral, T-REx™, and Gateway® technologies to facilitate easy recombination-
based cloning and lentiviral-based, regulated (Tetracycline-inducible), high-level
expression of a target gene in dividing and non-dividing mammalian cells. This
system is ideal expressing toxic proteins, because the inducible promoter allows the
control of the timing of gene expression.
• ViraPower™ Adenoviral Expression System is ideal for protein production,
and allows high-level transient gene expression in dividing and non-dividing
mammalian cells from the CMV or another promoter of choice. The ViraPower™
Adenoviral System uses Gateway® Technology for fast, easy, and accurate cloning of
the gene of interest.
For more information on ViraPower™ expression systems as well as on other expression

systems not discussed here, refer to www.lifetechnologies.com/proteinexpression.
Transient expression Stable expression

Viral
system Non-dividing Neuronal Growth- Contact-
Dividing cells Dividing cells
cells cells arrested cells inhibited cells
Adenovirus • •
Retrovirus • •
Lentivirus • • • • • •
75
Expression in insect cells Expression in insect cells offers significant advantages, including high expression levels,
ease of scale-up, and simplified cell growth that is readily adapted to high-density
suspension culture. Furthermore, because many of the posttranslational modification
pathways present in mammalian systems are also utilized in insect cells, proteins
produced in insect cells are antigenically, immunogenically, and functionally similar to
native mammalian proteins. Life Technologies™ offers powerful and versatile baculovirus
expression systems for high-level, recombinant protein expression in insect cells.
• BaculoDirect™ Baculovirus Expression System is a fast and easy method for

generating recombinant baculovirus using recombinational Gateway® cloning.
Baculovirus expression systems typically require bacterial transformation and
isolation of a large bacmid or co-transfection of a transfer vector and linear
baculovirus DNA into insect cells. The BaculoDirect™ system eliminates these time-
consuming steps, allowing the isolation of purified virus within one week. The
reduction of hand-on time for baculovirus generation makes the BaculoDirect™
system ideal for high-throughput expression.
• Bac-to-Bac® Baculovirus Expression System uses a unique bacmid shuttle vector
that recombines by site-specific transposition to generate an expression bacmid in
bacterial cells. The bacmid is then transfected into insect cells for the production of
recombinant baculovirus particles. With easy blue/white screening of recombinant
colonies, the Bac‑to‑Bac® Baculovirus Expression System is designed for fast, small
scale production of recombinant baculovirus.
• Bac-to-Bac® HBM Baculovirus Expression System enables secreted protein expression
via the honeybee melittin (HBM) secretion signal, which is ideal for proteins and
glycoproteins that require a secretion signal to be glycosylated. In contrast to
glycoproteins secreted from mammalian cells, glycoproteins secreted from baculovirus
can be easily de-glycosylated in vitro, which is essential for crystallizing the proteins.
• Bac-N-Blue™ Baculovirus Expression System is the classic and trusted expression
system for high-level recombinant protein production in insect cells. Recombinant
viral DNA is generated by co-transfection of a transfer vector containing the gene of
interest and the linear baculovirus DNA into insect cells. Recombinant baculovirus
is isolated using a blue/white plaque visualization method, and then amplified in
insect cells to generate a high-titer viral stock to initiate expression studies.
For more information on baculoviral expression systems as well as on other expression

systems not discussed here, refer to www.lifetechnologies.com/proteinexpression.
Secretion Fusion partner Expression/

System Host Promoter Advantage
signal Position Purif. Epitope inducer
BaculoDirect™ Sf9, Sf21, or — N-term 6×His V5 Polyhedrin Infection Fast and easy; ideal for
High Five™ C-term 6×His V5 high-throughput
Bac-to-Bac® or Sf9, Sf21, or Honeybee GST 6×His pFastBacHT Polyhedrin Infection Rapid baculovirus
Bac-to-Bac® HBM High Five™ melittin N-term pDEST10 or p10 production production; easy blue/
white selection
Bac-N-Blue™ Sf9, Sf21, or Honeybee C-term 6×His Xpress™ Polyhedrin Infection High-level
High Five™ melittin V5 recombinant protein
production
DES® S2 cells BIP C-term 6×His V5 MT or Ac5 CuSO4 or Constitutive or

constitutive inducible expression;
extremely high
integration

Guidelines for Plasmid DNA Transfection

Classic transfection technologies have initially been developed for introducing
plasmid DNA into cells, and plasmid DNA still remains the most common vector for
transfection. DNA plasmids containing recombinant genes and regulatory elements can
be transfected into cells to study gene function and regulation, mutational analysis and
biochemical characterization of gene products, effects of gene expression on the health
and life cycle of cells, as well as for large scale production of proteins for purification
and downstream applications.
The topology (linear or supercoiled) and the size of the vector construct, the quality
of the plasmid DNA, and the promoter choice are major factors that influence the
efficiency of plasmid DNA transfection.
Vector considerations Transient transfections are more efficient with highly supercoiled DNA compared to
linear DNA, presumably because circular DNA is not vulnerable to exonucleases, while
linear DNA fragments are quickly degraded by these enzymes (McLenachan et al.,
2007; von Groll et al., 2006). In addition, atomic force microscopy analysis shows very
different complexation patterns between cationic lipid reagents and circular and linear
DNA topologies: while compact spherical or cylindrical condensates are observed with
circular DNA, linear plasmids show extended pearl necklace-like structures. Although
the cationic lipid-mediated transfection of the more compact circular plasmids is likely
to go through endocytosis, the pathway of entry of extended linearized DNA structures
might be quite different and less efficient (von Groll et al., 2006).
Stable transfections are more efficient when using linear DNA due to its optimal
integration into the host genome. Linear DNA with free ends is more recombinogenic
and more likely to be integrated into the host chromosome to yield stable transformants,
even though it is taken up by the cell less efficiently.
Despite similar uptake efficiencies in cationic lipid-mediated transfection, nuclear

delivery of large plasmids is compromised compared with small plasmid molecules.
This effect is observed using equivalent mass or molar concentrations of different-sized
constructs, suggesting that nuclear delivery of plasmids may be limited by the rate of
intracellular transit and that small plasmids evade degradation by rapid transit through
the cytoplasm, rather than through the saturation of cellular defenses (Lukacs, et al.,
2000; McLenachan et al., 2007).
Quality of plasmid DNA Purity and quality of the plasmid DNA is critical for a successful transfection. The best
results are achieved with plasmid DNA of the highest purity that is free from phenol,
sodium chloride, and endotoxins. Contaminants will kill the cells, and salt will interfere
with lipid complexing, decreasing transfection efficiency. Endotoxins, also known as
lipopolysaccharides, are released during the lysis step of plasmid preparations and
are often co-purified with plasmid DNA. Their presence sharply reduces transfection
efficiency in primary and other sensitive cells. We recommend isolating DNA using
PureLink® HiPure Plasmid Kits (Mini, Midi, Maxi, Mega, and Giga), available from
Life Technologies™, that provide highest quality DNA for transfections. For more
information, refer to www.lifetechnologies.com/nap. Although cesium chloride
banding also yields highly purified DNA, it is a labor intensive and time consuming
process. Excess vortexing of DNA-lipid complexes or DNA solutions may result in some
shearing, especially with larger molecules, thereby reducing transfection efficiency. The
concentration of EDTA in the diluted DNA should not exceed 0.3 mM.
77
Gene product and

promoter Promoter choice is dependent on the host cell line, the protein to be expressed, and the
level of expression desired. Many researchers use the strong CMV (cytomegalovirus)
promoter because it provides the highest expression activity in the broadest range of
cell types. Another strong promoter for high-level protein expression in mammalian
cells is the EF-1α (human elongation factor-1£). However, using too strong a promoter
to drive the expression of a potentially toxic gene can cause problems in transient
transfection of plasmid DNA. For the potentially toxic gene products, use of weak
promoters are recommended
Toxic gene products are also a problem for selection of stably transfected cells. Cells
expressing a gene for antibiotic resistance lose their growth advantage when such gene
expression is detrimental to the health of the transfected cell, which makes it impossible
to obtain stably transfected clones with a constitutive promoter. In such cases, an
inducible promoter can be used to control the timing of gene expression, which will
allow for the selection of stable transfectants. Inducible promoters normally require the
presence of an inducer molecule (e.g., a metal ion, metabolite, or hormone) to function,
but some inducible promoters function in the opposite manner, that is, gene expression
is induced in the absence of a specific molecule.
Cell-type specific promoters, such as the polyhedrin promoter for insect cell
expression, are also common. Literature searches are the best tool to determine which
promoter will work best for your cell line or application.
Controls Regardless of the transfection method used, it is important to perform control

transfections to check for cell health, to determine whether the reported assay is
working properly, and to establish any insert-related problems. To check for optimal
cell growth conditions, include a negative control (no DNA, no transfection reagent). To
establish that the reporter assay is working properly, include a positive control (parallel
transfection with established transfection method). To determine whether there are
insert-related problems, transfect a plasmid without the gene of interest.
Optimization of Plasmid DNA Transfection

With any transfection procedure, a critical first step is to optimize the transfection
conditions. Every cell type and transfection procedure has a characteristic set of
requirements for optimal introduction of foreign DNA, and these conditions have a
large degree of variability even among cell types that are very similar to one another.
The single most important factor in optimizing transfection efficiency is selecting the
proper transfection protocol for the cell type. Once the appropriate transfection method
is selected, a transient reporter assay system can be used to optimize the procedure
by transfecting a reporter gene under a variety of conditions, and monitoring the
transfection efficiency by assaying for the reporter gene product.
This section provides general guidelines for optimizing calcium phosphate–mediated

gene transfer, electroporation using the Neon® Transfection System, and cationic lipid-
mediated transfection.

Considerations for
calcium phosphate
co‑precipitation The primary factors that influence the efficiency of calcium phosphate transfection are
the amount of DNA in the calcium-phosphate–DNA co-precipitate, the length of time
the cell is incubated with the co-precipitate, and the use and duration of glycerol or
DMSO shock.
Total DNA amount used in calcium phosphate transfection is usually 10–50 μg in 450 μL
sterile water and 50 μL of 2.5 M CaCl2 per 10‑cm dish, but varies widely among plasmid
preparations as well as with different cells and media. While with some cell lines
10–15 μg of DNA added to a 10-cm dish results in excessive cell death and very little
uptake of DNA, other cell lines, especially primary cells, much higher concentrations
of DNA is required. Each new plasmid preparation and each new cell line being
transfected should be tested for optimum DNA concentration.
The optimal length of time that the cells are incubated with co-precipitate also varies
with cell type. Some hardy cell types, such as HeLa, NIH 3T3, and BALB/c 3T3, are
efficiently transfected by leaving the co-precipitate on for up to 16 hours, which might
kill some more sensitive cells.
A pilot experiment varying the amount of DNA, incubation time, and exposure to
glycerol or DMSO shock will indicate whether the cell type is tolerant to long exposure
to a calcium phosphate precipitate and whether glycerol shock should be used. Once
the results of the pilot experiment are obtained, further optimization can be performed
by adjusting the experimental variables even finer. For instance, if shocking the cells
with 10% glycerol for 3 minutes as shown in the example below enhances transfection
efficiency, an experiment varying the time of glycerol shock or using 10–20% DMSO
shock might also be tried.
Dish (10-cm) Reporter plasmid (μg) Incubation (hour) Glycerol shock (minutes)
1 5 6 —
2 10 6 —
3 15 6 —
4 20 16 —
5 25 16 —
6 30 16 —
7 5 6 3
8 10 6 3
9 15 6 3
10 20 16 3
11 25 16 3
12 30 16 3
Figure 6.2 Pilot experiment example for the optimization of transfection by calcium phosphate
co‑precipitation.
79
Considerations for
cationic lipid-mediated
delivery Four primary parameters affect the success of DNA transfection by cationic liposomes:
the amount of DNA, the ratio of transfection reagent to DNA, incubation time of the
lipid-DNA complex, and the cell density at the time of complex addition. These factors
should be systematically examined for every cell type and vector combination, and once
optimized, kept constant in all future experiments to help ensure reproducible results.
For best results, follow the optimization protocols provided by the manufacturers of the
reagent. Life Technologies™ provides optimization protocols for all of its transfection
reagents. For more information, refer to www.lifetechnologies.com/transfection.
Amount of DNA
The optimal amount of DNA varies depending on the characteristics of the transfected
plasmid (e.g., promoter, size of plasmid, origin of replication), number of cells to be
transfected, size of the culture dish, and the target cell line used. In many of the cell
types tested, relatively small amounts of DNA are effectively taken up and expressed. In
fact, higher levels of DNA can be inhibitory in some cell types with certain cationic lipid
preparations. In addition, cytotoxicity may result if a plasmid encoding a toxic protein
or too much plasmid with a high expression rate is used.
Ratio of transfection reagent to DNA

The overall charge of the transfection complexes is determined by the ratio of transfection
reagent to DNA. The negative charge contributed by phosphates within the DNA
backbone needs to be offset by the positive charge contributed from the transfection
reagent for both good complex formation and for neutralizing the electrostatic repulsion
imparted on the DNA by the negatively charged cell membrane.
The optimal ratio of transfection reagent to DNA is highly cell type-dependent. As a

starting point, the amount of transfection reagent should be varied while keeping a
constant plasmid DNA concentration (for example, 1:1, 3:1 and 5:1 ratios of volume to
mass). Additional benefits may be derived by maintaining the ratio and increasing the
amount of plasmid added.
Incubation time
The optimal incubation period of cells with the transfection complexes depends on
the cell line and transfection reagent used. In general, transfection efficiency increases
with time of exposure to the lipid reagent-DNA complex, although toxic conditions
can develop with prolonged exposure to certain lipid reagents, requiring removal
by centrifugation or dilution with fresh medium after a given incubation period to
minimize cytotoxic effects. However, newer and gentler transfection reagents such as
the Lipofectamine® 3000 reagent do not necessitate complex removal or dilution after
transfection (see www.lifetechnologies.com/3000 for more information).
When using cationic lipid reagents that require adding or replacing the medium, vary the
incubation time after complex addition (e.g., 30 minutes to 4 hours, or even overnight)
and monitor cell morphology during the this interval, particularly if the cells are
maintained in serum-free medium as some cell lines lose viability under these conditions.

Cell density
Cell density also affects overall transfection efficiency. To achieve transcription and
ultimately protein production, nuclear deposition of DNA is required, which is largely
dependent on membrane dissolution and reformation during mitosis, requiring that the
cells have to be actively dividing.
For adherent cells, the best efficiency is often attained at a confluency of 80%, but
protocol recommendations may range from 40–90%. For suspension cells, we
recommend splitting the cells the day prior to transfection to ensure that the cells
will be in optimal physiological condition for the transfection procedure. The optimal
density is highly dependent on cell type and reagent-specific toxicity, and should be
determined empirically.
Timeline Steps
Seed cells to be 70–90% confluent

Day 0
1 at transfection
Diluted Lipofectamine® 3000
Dilute Lipofectamine® 3000

2 Reagent in Opti-MEM® Medium
Vortex 2–3 sec
Diluted DNA
Prepare master mix of DNA
by diluting DNA in Opti-MEM®
3 Medium, then add P3000™ Reagent
– Mix well
Add Diluted DNA to each tube

4
Day 1
of Diluted Lipofectamine® 3000

Reagent (1:1 ratio)
5 Incubate
6 Add DNA-lipid complex to cells

Day 2–4
7 Visualize/analyze transfected cells
Figure 6.3 Example transfection workflow using the Lipofectamine® 3000 transfection reagent.
81
Considerations for
electroporation Electroporation is mainly dependent on the combination of three electric parameters:
the pulse voltage, pulse width, and pulse number. Perhaps because it is not a chemically
based protocol, electroporation is less affected by DNA concentration; however, it
requires almost five-fold more cells and DNA compared to calcium phosphate‑mediated
transfection. Generally, 1–5 μg of DNA per 107 cells is sufficient, and there is a good
linear correlation between the amount of DNA present and the amount taken up.
The objective in optimizing electroporation parameters is to find a pulse that maintains

40–80% survival of the cells. The pulse width is determined by the capacitance of the
power source and the extent to which this can be varied depends on the electronics of
the power supply generating the pulse. If excessive cell death occurs, the length of the
pulse can be lowered by lowering the capacitance.
Keeping cells on ice often improves cell viability and results in higher effective
transfection frequency, especially at high power which can lead to heating (Potter
et al., 1984). However, some cell lines electroporate with higher efficiency at room
temperature under low voltage/high capacitance conditions (Chu et al., 1991).
The Neon® Transfection System, available from Life Technologies™, is pre-programmed

with 18- and 24-well optimization protocols that allow quick optimization of electric
parameters for many adherent and suspension cell lines within days. Cell line-specific
optimized protocols for the Neon® Transfection System can also be conveniently
downloaded from www.lifetechnologies.com/neon to maximize transfection
efficiencies for many commonly used cell types.

Selection of Stable Transfectants

Selection of stably transfected cells begins with successful transient transfection with
a plasmid containing a selectable marker, such as an antibiotic resistance gene. As
a negative control, cells should be transfected using DNA that does not contain the
selectable marker.
Before starting • Ensure that the cell line you are using can produce colonies from isolated cells as
some cells require contact with one another to grow. For such cells, adapted or
conditioned medium may be beneficial.
• Choose an appropriate selectable marker (see Selection Antibiotics for Eukaryotic
Cells, page 62).
• Select a transfection procedure suitable for your cell type.
• Determine the selective conditions for your cell type by establishing a dose-response
curve (kill curve) (Ausubel et al. 1995).
Kill curve A kill curve should be established for each cell type and each time a new lot of the
selective antibiotic is used.
1. Split a confluent dish of cells at approximately 1:5 to 1:10 (depending on the cell type
and cell density post-transfection) into medium containing various concentrations of
the antibiotic.
2. Incubate the cells for 10 days replacing selective medium every 4 days (or as needed).
3. Examine the dishes for viable cells using the desired method (e.g., Countess® II
Automated Cell Counter, hemocytometer with trypan blue staining).
4. Plot the number of viable cells versus antibiotic concentration to establish a kill curve
to determine the most appropriate selective drug concentration required to kill
untransfected cells.
Selection workflow 1. Transfect the cells using the desired transfection method. If the selectable marker is on a
separate vector, use a 5:1 to 10:1 molar ratio of plasmid containing the gene of interest to
plasmid containing the selectable marker.
Note: Perform control transfections with a vector containing the selectable marker but
not the gene of interest. If colonies are obtained from cells transfected with the control
plasmid but not from cells transfected with plasmid containing the gene of interest,
indicating that the gene of interest may be toxic. It is also important to perform replicate
transfections in case the transfection fails or the cultures become contaminated.
2. Forty-eight hours after transfection, passage the cells at several different dilutions
(e.g., 1:100, 1:500) in medium containing the appropriate selection drug. For effective
selection, cells should be subconfluent, because confluent, non-growing cells are
resistant to the effects of antibiotics like Geneticin®. Suspension cells can be selected in
soft agar or in 96-well plates for single-cell cloning.
83
3. For the next two weeks, replace the drug-containing medium every 3 to 4 days (or as
needed).
Note: High cell densities in suspension cultures require frequent medium changes
that may deplete critical soluble growth factors, thereby reducing cell viability and the
efficiency of the system.
4. During the second week, monitor cells for distinct “islands” of surviving cells.
Depending on the cell type, drug‑resistant clones will appear in 2–5 weeks. Cell death
should occur after 3–9 days in cultures transfected with the negative control plasmid.
5. Isolate large (500–1,000 cells), healthy colonies using cloning cylinders or sterile
toothpicks, and continue to maintain cultures in medium containing the appropriate
drug (for the isolation of clones in suspension culture, see Freshney, 1993).
6. Transfer single cells from resistant colonies into the wells of 96-well plates to confirm
that they can yield antibiotic-resistant colonies. Ensure that only one cell is present per
well after the transfer.

Selecting a RNAi Strategy

The two common approaches for RNAi delivery are lipid-mediated transfection and
viral-mediated transduction. Determining which one of these approaches to use
depends on the cell type being studied and whether transient or stable knockdown is
desired. The most popular application, transient transfection of Silencer® Select siRNAs
or Stealth RNAi™ siRNA duplexes, uses cationic lipid–based reagents because they are
suitable for delivering molecules across a diverse range of commonly used cell lines (see
Non-vector siRNA technologies, page 86)
For cell types that are not amenable to lipid-mediated transfection, viral vectors are
often employed (see Vector-mediated RNAi, page 89). Adenoviral vectors work well
for transient delivery in many cell types. However, when stable RNAi expression is
desired, or for difficult cell lines, such as nondividing cells, lentiviral vectors are the best
delivery method. Another approach for determining the most favorable RNAi delivery
conditions is to use Life Technologies™ delivery optimization service—a scientific
resource with extensive knowledge and expertise in viral vectors and non-viral delivery
reagents for testing a matrix of delivery parameters.
siRNA vs. vector

approaches Both siRNA and vector-based RNAi can be extremely effective at producing loss of
function phenotypes. In general, most researchers choose siRNA because they can
start quickly and there are no special preparations needed other than basic cell culture
techniques. However, there are a number of reasons why a researcher might choose either
siRNA or a vector-based RNAi.
Typically, researchers strive to achieve the highest levels of transfection efficiency

possible. This objective is particularly important for RNAi applications because
non‑transfected cells will continue to express the gene targeted for knockdown, thus
contributing to background expression levels.
For many disease models, the most desirable cell types are primary cultures. However,
these cannot be transfected adequately with commercially available cationic lipid-mediated
transfection reagents. A powerful alternative is viral delivery of vectors expressing RNAi
sequences. This option is recommended for delivery to hard-to-transfect, primary, and
nondividing cells. Viral delivery can also be used to create stable cell lines with inducible
RNAi expression or to express RNAi sequences with tissue-specific promoters.
Transient expression
Cell type Stable expression
(<7 days) (>7 days)
Fast-growing adherent Lipid transfection of Silencer® Select Lipid transfection of RNAi vectors or Lipid transfection of RNAi
cells (A549, HeLa) siRNA or Stealth RNAi™ siRNA adenoviral delivery vectors or lentiviral delivery
Lipid transfection or electroporation Lipid transfection or

Fast-growing suspension Lipid transfection of RNAi vectors or
of Silencer® Select siRNA or Stealth electroporation of RNAi vectors
cells (THP-1) adenoviral delivery
RNAi™ siRNA or lentiviral delivery
Primary cells Lentiviral delivery
Nondividing cells Lentiviral delivery
85
Non-vector siRNA
technologies For transient knockdown experiments, synthetic, non-vector approaches offer significant
advantages over vector-based methods for RNAi delivery. In particular, nonvector
experiments are typically easier to design and perform and can result in higher levels
of transient knockdown. In addition, recent improvements in RNAi design have
increased the likelihood of achieving high-level knockdown after testing only a few
RNAi molecules. Consequently, using synthetically generated RNA duplexes is the most
popular method for conducting RNAi experiments.
Synthetic siRNAs
Traditional RNAi methods for gene knockdown in mammalian cells involved the use
of synthetic RNA duplexes consisting of two unmodified 21-mer oligonucleotides
annealed together to form short/small interfering RNAs (siRNAs). Life Technologies™’
Silencer® Select siRNA and Stealth RNAi™ siRNA improve upon these traditional
duplexes by using proprietary chemical modifications to ensure better RNAi results.
For more information, see www.lifetechnologies.com/sirna.
• Silencer® siRNAs are Ambion®-designed siRNAs available for all human, mouse,
and rat gene targets in the RefSeq database. These siRNAs are designed for
maximum potency and specificity using a highly effective and extensively tested
algorithm. Each siRNA is synthesized to the highest quality standards and is
provided with full sequence information.
• Stealth RNAi™ siRNA molecules are chemically modified, blunt-ended, 25-mer
double-stranded duplexes that are recognized by the RNA-induced silencing
complex (RISC) to mediate inhibition of a target gene. Proprietary chemical
modifications allow Stealth RNAi™ siRNA to overcome many in vivo–specific
obstacles, allowing effectiveness and stability in in vivo applications.
• Silencer® Select siRNAs are the best-performing siRNAs for in vitro studies, and are
available in a variety of formats including preplated collections and custom libraries
to simplify screening experiments. They are up to 100-fold more potent than other
siRNAs (modified and unmodified), allowing a higher percentage of “on-target”
phenotypes.
Silencer® siRNA Stealth RNAi™ siRNA Silencer® Select siRNA
Potency 100 nM recommended concentration 20 nM recommended concentration 5 nM recommended concentration
Target specificity Moderate High Highest
Innate immune Minimized through chemical Minimized through chemical

Low
response modifications modifications
Unmodified 21-bp duplex with Modified 25-bp duplex with no LNA modified 21-bp duplex with
Molecular format
overhangs overhangs overhangs
Coverage Coding RNA Coding RNA Coding and non-coding RNA
Human, mouse, rat Human, mouse, rat Human, mouse, rat

Target species
(Other species: use custom tool) (Other species: use custom tool) (Other species: use custom tool)
Custom design tool Custom Silencer® siRNA Custom Stealth RNAi™ siRNA Custom Silencer® Select siRNA
Good knock-down, low off-target Highest knock-down, lowest off-target

Notes Cost-effective siRNA
effects effects

miRNA mimics and inhibitors

Analyses of miRNA function are performed using strategies that are similar to those
used for protein-encoding genes. Transfecting cultured cells with miRNA mimics can
help identify gain-of-function phenotypes; down-regulation or inhibition experiments
using miRNA inhibitors can be conducted to identify loss-of-function phenotypes. The
combination of up-regulation and down-regulation can be used to identify genes and
cellular processes that are regulated by specific miRNAs.
• Ambion® Pre-miR™ miRNA Precursors are small, chemically-modified double-

stranded RNA molecules that are similar, but not identical, to siRNAs, and are
designed to mimic endogenous mature miRNAs.
• mirVana™ miRNA Mimics are small, chemically modified double-stranded
RNAs that mimic endogenous miRNAs and enable miRNA functional analysis
by up-regulation of miRNA activity. These molecules are more specific than
their predecessors due to inactivation of the star strand by proprietary chemical
modifications. mirVana™ miRNA Mimics are available individually or as libraries
• Ambion® Anti-miR™ miRNA Inhibitors are chemically modified, single-stranded
nucleic acids designed to specifically bind to and inhibit endogenous miRNAs.
• mirVana™ miRNA Inhibitors are small, chemically modified single-stranded RNA
molecules designed to specifically bind to and inhibit endogenous miRNA molecules
and enable miRNA functional analysis by down-regulation of miRNA activity.
They have the highest potency in vitro inhibition at the lowest miRNA inhibitor
concentration of available miRNA mimics. mirVana™ miRNA Inhibitors are available
individually or as libraries.
Due to their small size, these synthetic molecules are easier to transfect than vectors,
and can be delivered using conditions identical to those used for siRNAs. In contrast to
miRNA expression vectors, they can also be used in dose response studies.
Note: Pre-miR miRNA Precursors are not hairpin constructs and should not be
confused with pre-miRNAs.
Ambion® Pre-miR™ Ambion® Anti-miR™ mirVana™ miRNA

mirVana™ miRNA Mimics
Precursors Inhibitors Inhibitors
Function Mimic endogenous miRNAs Mimic endogenous miRNAs Inhibit endogenous miRNAs Inhibit endogenous miRNAs
Experimental Gain of function Gain of function Loss of function Loss of function

objective (in vivo or in vitro) (in vivo or in vitro) (in vivo or in vitro) (in vivo or in vitro)
100% Coverage of 100% Coverage of 100% Coverage of 100% Coverage of

Content database
miRBase v 15* miRBase v 19† miRBase v 15* miRBase v 19†
Model system in vitro in vitro & in vivo in vitro in vitro & in vivo
Human, mouse, rat Human, mouse, rat

Species covered Human (Other species: use custom Human (Other species: use custom
tool) tool)
GeneAssist™ miRNA GeneAssist™ miRNA GeneAssist™ miRNA GeneAssist™ miRNA

Custom design tool
Workflow Builder Workflow Builder Workflow Builder Workflow Builder
Pre-Designed or Custom Pre-Designed or Custom Pre-Designed or Custom Pre-Designed or Custom

miRNA libraries
Libraries Libraries Libraries Libraries
Novel design to minimize Next generation chemistries Chemically modified for Next generation chemistries
Notes
off-target effects for lowest off-target effects good efficacy for highest efficacy
* 1090 distinct human sequences; † 2019 distinct human sequences.
87
siRNA transfection siRNAs are easily introduced into cells with a siRNA transfection reagent . Soon after
being inserted in the mammalian cell, the siRNA molecules become a part of the
RNA‑induced silencing complex (RISC). Guided by the antisense strand of the siRNA,
RISC degrades the targeted mRNA inhibiting its translation. Assays are then performed
to detect the RNAi activity. Controls are normally set up so RNAi results can be
properly compared.
The success of RNAi is dependant on correct delivery of siRNA in appropriate amount

at a time when it will brings about the maximum expected response. Such precision can
be tricky. Off-targeting by siRNAs proves lethal and poses analytical issues at times.
Researchers are looking for better ways of designing and delivering siRNA
Transfection
Transfection method Payload Cell viability Notes
efficiency
Most efficient versatile reagent for the

Superior Superior widest range of cell types including
reagent
difficult-to-transfect cells.
Lipofectamine® 2000 High efficiency versatile reagent for a

High High
reagent wide range of common cell types.
Lipofectamine® RNAiMAX Most efficient reagent for siRNA/miRNA

Superior Superior
reagent delivery. Efficient gene knock-down.
High-efficiency electroporation for all cell

Neon® electroporation Maximal Good
lines.

mRNA for expression
of protein

Vector-mediated RNAi For cell types not amenable to lipid-mediated transfection, such as hard‑to-transfect,
primary, and non-dividing cells, viral vectors containing RNAi cassettes are often
employed. Viral delivery can also be used to create stable cell lines with inducible RNAi
or to express RNAi sequences with tissue-specific promoters. Adenoviral vectors work
well for transient delivery in many cell types, while lentiviral vectors are best for stable
delivery in dividing and non‑dividing cells, lentiviral vectors are best.
• BLOCK-iT™ Adenoviral RNAi Expression System facilitates the creation and

delivery of a replication-incompetent adenovirus to transiently express shRNA in
most dividing or non‑dividing mammalian cell types and animal models for RNAi
analysis. The key advantage of the BLOCK-iT™ Adenoviral RNAi Expression System
is Gateway® recombination technology, which simplifies the cloning and generation
of an adenoviral vector, eliminating the tedious and time-consuming manipulations,
screening, and multiple transformations that other adenoviral systems require
• BLOCK-iT™ Lentiviral RNAi Expression System enables the creation and delivery
of engineered shRNAs and miRNAs into dividing and non‑dividing mammalian
cells, including primary and hard-to-transfect cells. The system can be used without
selection for transient RNAi analysis or, with appropriate antibiotic selection, to
generate a stable cell line for long-term knockdown studies.
• BLOCK-iT™ Lentiviral Pol II miR RNAi Expression System combines BLOCK-iT™
Pol II miR RNAi and ViraPower™ Lentiviral technologies to facilitate the creation
and stable delivery of engineered miRNAs into nondividing, primary, and hard-
to-transfect cells. The Pol II promoter in the expression vector enables co-cistronic
expression of multiple miRNAs, allowing knockdown of multiple targets from
a single construct, a process is ideal for knockdown of more than one pathway
component or splice variant, or for using knockdown to create synthetic phenotypes.
• BLOCK-iT™ Lentiviral Pol II miR RNAi Expression System with EmGFP provides
all the components and benefits of the BLOCK-iT™ Lentiviral Pol II miR RNAi
Expression System listed above, plus the easy expression tracking with co-cistronic
EmGFP. The HiPerform™ version of the expression vector contains an mRNA-
stabilizing sequence (WPRE) and a nuclear import sequence (cPPT) that can generate
up to 5-fold higher virus titers.
• BLOCK-iT™ Inducible H1 Lentiviral RNAi System is a complete lentiviral system
for long-term inducible or constitutive shRNA expression in any cell type. Regulation
of the RNAi response via the tetracycline operator (TetO2) sequence permits the
study of changes over time and loss-of-function experiments even with essential
genes, and provides an excellent control system to measure phenotypic changes
during recovery of gene function.
Viral system When to use
• High-level transient shRNA expression

Adenoviral RNAi delivery • Effective delivery to a wide range of human cell types
• Studies in animal models
• Stable expression of RNAi in any cell line, even in non‑dividing cells

including stem cells, lymphocytes, and neurons
Lentiviral RNAi delivery
• Inducible or constitutive shRNA or miR RNAi expression
• Studies in animal models
89
Guidelines for RNA Transfection

Transfection of RNA is an offshoot of classic transfection technologies for introducing
RNA into cells. The purpose of RNA transfection is similar to that of plasmid
transfection. mRNA is introduced into cells to express the encoded protein, and study
gene function and regulation. siRNA is used for RNAi studies that examine the effects
of gene knockdown. One major difference between the two methods is that RNA can
only be transiently transfected.
RNAi workflow The diagram below depicts an RNAi experiment workflow following siRNA design
and synthesis. When performing an RNAi experiment, make sure that you have the
following on hand:
• Transfection/electroporation agent and protocol

• Assays to assess knockdown and other RNAi effect(s)
• Positive and negative control siRNAs
• Two or more siRNAs to gene of interest
1 Find and order siRNAs at:

www.lifetechnologies.com/RNAi
2 Plate cells and

transfect siRNAs
3 Prep RNA
t
3' 5'
5' 3'
4 Monitor siRNA-induced knockdown to: 4 Observe/measure phenotypic change

• Validate the siRNA
• Monitor transfection efficiency
100
10
% RNA remaining
80
Control siRNA
60
1
Rn
40
0.1
30
20
Surviving siRNA
0.01
0 0 5 10 15 20 25 30 35 40
RPS6KA4-1
RPS6KA4-2
RPS6KA4-3
MAP2K1-1
MAP2K1-2
MAP2K1-3
MAP2K4-1
MAP2K4-2
MAP2K4-3
PCNA gold
TGFBR2-1
TGFBR2-2
TGFBR2-3
STK12-1
STK12-2
STK12-3
PK428-1
PK428-2
PK428-3
NEK4-1
NEK4-2
NEK4-3
STK6-1
STK6-2
WEE1-1
WEE1-2
WEE1-3
STK6-3
Cycle
Figure 6.4 RNAi workflow following siRNA design and synthesis.

Handling RNA RNA oligonucleotides are susceptible to degradation by exogenous ribonucleases

introduced during handling.
• Wear gloves when handling RNA.

• Use RNase-free reagents, tubes, and barrier pipette tips for preparing RNA for
transfection.
• Work areas should be wiped down with 70% ethanol or other RNase-
decontamination solution such as RNaseZap® RNase Decontamination Solution.
Transfection efficiency The efficiency with which mammalian cells are transfected with siRNA will vary
according to cell type and the transfection agent used. This means that the optimal
concentration used for transfections should be determined empirically. The major
variables that impact siRNA transfection efficiency are the following:
• Transfection reagent type and amount

• Number of cells plated in well
• Type of RNA or siRNA
• Concentration of RNA or siRNA.
Positive controls It is important to include a positive control in each experiment. The positive control
should elicit a reproducible, easily measured response in the cells and assay used in
your study. If you see maximal effect above/below a pre-determined threshold level
with this control, you know that measurements from other experiments tested on the
same day are reliable. Note that it is important to empirically determine the thresholds
for each assay and control pair.
The degree of the response to a particular RNA or siRNA is directly linked to its
transfection efficiency. To assess transfection efficiency, we recommend including the
BLOCK-iT™ Fluorescent Oligo in every experiment. Using the BLOCK-iT™ Fluorescent
Oligo in your transfection experiment allows you to easily assess oligomer uptake and
transfection efficiency using any fluorescence microscope and a standard FITC filter
set. Uptake of the fluorescent oligomer by at least 80% of cells correlates with high
efficiency.
Negative controls Negative controls are just as important as positive controls for obtaining meaningful
data. Always include a set of transfections with an equimolar amount of at least one
negative control to compare the effects of the target RNA or siRNA-treated and control-
treated cells. Data from these crucial controls serve as a baseline for evaluation of
experimental target knockdown.
Non-transfected or cells-only negative controls are also very useful. By comparing

expression of a housekeeping gene among cultures that were not transfected and
cultures transfected with a non-targeting negative control, valuable information about
the effects of transfection on cell viability can be obtained.
91
Type of control Recommended use Recommended products

• BLOCK-iT Alexa Fluor® Red Fluorescent Control
™
Transfection control Calculate and monitor transfection efficiency with fluorescence

• BLOCK-iT™ Fluorescent Oligo
• Silencer® Select Negative Control siRNAs
Nonspecific or scrambled controls used to measure knockdown
Negative controls • Stealth RNAi™ siRNA negative controls
levels vs. background
• Silencer® negative control siRNAs
• Silencer® Select GAPDH Positive Control siRNAs
RNAi reagents known to achieve high levels of knockdown used
Positive controls • Stealth RNAi™ siRNA positive controls
to measure delivery and optimize experimental conditions
• Silencer® positive control siRNAs
Untransfected
Measure normal gene expression level and phenotype
control
Multiple RNAi
Use to verify phenotypic change, control for off-target effects
sequences to the
for generating publication quality results
same target
Use the lowest effective level to avoid altering the cells normal
Titration of RNAi
processes
BLOCK-iT™ Pol II miR RNAi or BLOCK-iT™ shRNA
Turn off inducible RNAi or introduce a plasmid expressing the
Rescue experiments vectors with inducible promoters (CMV/TO and H1/TO
target mRNA that the RNAi sequence will not affect
respectively)
Co-transfection Co-transfection is performed when the user wants to introduce both siRNA and a
plasmid for expressing a protein into a cell. This protein can be part of the test system,
or in most cases, it can be a reporter gene (luciferase, GFP, β-lactamase). In some cases,
users may want to express a mutant protein along with the siRNA to block one pathway
with the siRNA, and overexpress a mutant protein.
The presence of the plasmid may decrease transfection efficiency of all cargo
(plasmid and siRNA) when a lipid transfection reagent is used, making transfection
optimizations very important. Undesired and non-specific cell death can result with too
much lipid, or too little knock-down or protein expression from the plasmid can occur if
transfection conditions are not optimal.
siRNA quality The quality of siRNA can significantly influence RNAi experiments. siRNAs must
be free of reagents carried over from synthesis, such as ethanol, salts, and proteins.
Also, dsRNA contaminants longer than 30 bp are known to alter gene expression by
activating the nonspecific interferon response and causing cytotoxicity (Stark et al.,
1998). Therefore, we recommend using standard purity siRNAs that are greater than
80% full length.
siRNA storage
Store siRNAs at –20°C or –80°C, but do not use a frost-free freezer. Our data indicate
that up to 50 freeze/thaw cycles are not detrimental to siRNAs in solution at 100 μM (as
assessed by mass spectrometry and analytical HPLC). However, we recommend that
siRNAs that have been resuspended in RNase-free water or buffer be stored in small
aliquots to avoid potential contamination.
Nuclease resistance of siRNAs

Annealed, double-stranded siRNAs are much more nuclease resistant than single-
stranded RNA. However, stringent RNase-free techniques should be used during all
RNAi experiments.

Checking siRNA for degradation

If you suspect that a preparation of siRNA may be degraded, check the integrity of the
siRNA by running ~2.5 μg on a non-denaturing 15–20% acrylamide gel. Visualize the
RNA by staining with ethidium bromide, and verify that it is the expected size and
intensity. The siRNA should migrate as a tight band; smearing indicates degradation.
siRNA quantity The optimal amount of siRNA and its capacity for gene silencing are influenced in part
by properties of the target gene products, including the following: mRNA localization,
stability, abundance, as well as target protein stability and abundance.
Although many siRNA experiments are still performed by transfecting cells with
100 nM siRNA, published results indicate that transfecting lower siRNA concentrations
can reduce off-target effects exhibited by siRNAs (Jackson et al., 2003; Semizarov et al.,
2003). For lipid-mediated reverse transfections, 10 nM of siRNA (range 1–30 nM) is
usually sufficient. For siRNA delivery using electroporation, siRNA quantity has a less
pronounced effect, but typically 1 μg/50 µL cells (1.5 μM) of siRNA (range
0.5–2.5 μg/50 µL cells or 0.75–3.75 μM) is sufficient.
Keep in mind that while too much siRNA may lead to off-target or cytotoxic effects,
too little siRNA may not reduce target gene expression effectively. Because there are so
many variables involved, it is important to optimize the siRNA amount for every cell
line used. In addition, the amount of non-targeting negative control siRNA should be
the same as the experimental siRNAs.
Volume of transfection
reagent The volume of transfection agent is a critical parameter to optimize because too little
can limit transfection, and too much can be toxic. The overall transfection efficiency is
influenced by the amount of transfection agent complexed to the siRNA. To optimize,
titrate the transfection agent over a broad dilution range, and choose the most dilute
concentration that still gives good gene knockdown. This critical volume should be
determined empirically for each cell line.
Cell density While cell density is important for traditional, pre-plated transfection experiments, cell
density is less critical and requires little to no optimization, when siRNAs are delivered
by reverse transfection. However, if too many cells are used, and the amount of siRNA
is not increased proportionally, the concentration of siRNA in the sample may be
too low to effectively elicit gene silencing. When cell density is too low, cultures can
become unstable. Instability can vary from well to well because culture conditions (e.g.,
pH, temperature) may not be uniform across a multiwell plate and can differentially
influence unstable cultures.
Exposure to transfection
agent/siRNA complexes Although most transfection agents are designed to induce minimal cytotoxicity,
exposing cells to excessive amounts of transfection agent or for an extended time can be
detrimental to the overall health of the cell culture. Sensitive cells may begin to die from
exposure to the transfection agent after a few hours. If transfection causes excessive cell
death with your cells, remove the transfection mixture and replenish with fresh growth
medium after 8–24 hours.
93
Presence of serum
during transfection Complex formation between transfection agents and siRNA should be performed in
reduced-serum or serum-free medium, so that serum components will not interfere
with the reaction. However, once complex formation has occurred, some transfection
agents will permit transfection in serum-containing, normal growth medium (follow
manufacturer’s instructions). No culture medium addition or replacement is usually
required following transfection, but changing the media can be beneficial in some
cases, even when serum compatible reagents are used. Be sure to check for serum
compatibility before using a particular agent. Some transfection agents require serum-
free medium during the transfection and a change to complete growth media after an
initial incubation with transfection complexes.
Tips for a successful

siRNA experiment 1. Design and test two to four siRNA sequences per gene. Do not attempt to design
siRNAs on your own. Go to www.lifetechnologies.com/rnai and utilize the best-in-
class design algorithms to design your siRNAs.
2. Avoid RNases! Trace amounts of ribonucleases can sabotage siRNA experiments. Since
RNases are present throughout the laboratory environment on your skin, in the air, on
anything touched by bare hands or on anything left open to the air, it is important to
take steps to prevent and eliminate RNase contamination. Life Technologies™ offers a
complete line of products designed to detect and eliminate RNases.
3. Maintain healthy cell cultures and strict protocols for good transfection reproducibility.
In general, healthy cells are transfected at higher efficiency than poorly maintained
cells. Routinely subculturing cells at a low passage number ensures that there will
be minimal instability in continuous cell lines from one experiment to the next.
When performing optimization experiments we recommend transfecting cells within
50 passages, since transfection efficiency drops over time.
4. Avoid antibiotic use. Avoid the use of antibiotics during plating and up to 72 hours
after transfection. Antibiotics have been shown to accumulate to toxic levels in
permeabilized cells. Additionally, some cells and transfection reagents require serum-
free conditions for optimal siRNA delivery. We suggest you perform a pilot transfection
experiment in both normal growth media and serum-free media to determine the best
condition for each transfection.
5. Transfect siRNAs using optimized reagents. Use an optimized siRNA transfection

reagent and protocol for your cell type. The choice of transfection reagent is critical for
success in siRNA experiments. It is essential to use transfection reagents formulated to
deliver small RNAs (most commercially available transfection reagents were designed
for large plasmid DNA, not small RNA molecules). Also, some reagents have been
developed for the transfection of specific cell lines while others have broader specificity.
For help selecting the appropriate transfection reagent, see siRNA transfection,
page 88.
6. Use an appropriate positive control to optimize transfection and assay conditions.

Housekeeping genes are suitable positive controls for most cell types. To optimize
conditions, transfect target cells with several concentrations of an siRNA specific to
your chosen positive control and to your experimental target siRNA. Measure the
reduction in the control protein or mRNA level compared to untransfected cells 48
hours after transfection. Too much siRNA can lead to cell toxicity and death. For
maximum convenience, Life Technologies™ offers positive control siRNAs against a
variety of gene targets (see page 92).

7. Use a negative control siRNA to distinguish non-specific effects (see page 92).
Negative controls should be designed by scrambling the nucleotide sequence of the
most active siRNA. However, be sure to perform a homology search to ensure that your
negative control sequence lacks homology to the genome of the organism being studied.
8. Use labeled siRNAs for protocol optimization. Fluorescently labeled siRNA can be
used to analyze siRNA stability and transfection efficiency. Labeled siRNA is also useful
to study siRNA subcellular localization and in double label experiments (with a labeled
antibody) to visualize cells that receive siRNA during transfection and to correlate
transfection with down-regulation of the target protein.
Optimization of siRNA Transfection

Factors affecting siRNA
transfection efficiency Maximizing transfection efficiency while minimizing cytotoxicity are crucial for optimal
gene silencing. Similar to balancing siRNA-induced knockdown and cell viability, there
may also be a balance between siRNA delivery and downstream phenotypic assay
conditions. It may be necessary to re-optimize siRNA delivery conditions for different
downstream assays that are used in siRNA screening passes. The best transfection
efficiencies are achieved for each cell type by identifying the following factors (in order
of importance):
1. Choice of transfection reagent
2. Volume of transfection agent
3. Amount of siRNA
4. Cell density at the time of transfection
5. Length of exposure of cells to transfection agent/siRNA complexes
6. Transfection method: traditional transfection where cells are pre-plated or reverse

transfection where cells are transfected as they adhere to the plate
7. Presence or absence of serum
Once the conditions for maximal gene silencing are determined, keep them constant
among experiments with a given cell type.
95
Appendix
Troubleshooting
The table below lists some potential problems and possible solutions that may help
you troubleshoot your cell culture experiments. Note that the list below includes only
the most commonly encountered problems in cell culture, and provides guidelines to
solutions only. To help evaluate your results more successfully, we recommend that you
consult the manuals and product information sheets provided with the products you
are using as well as the published literature and books on the subject.
Problem Reason Solution

No viable cells after thawing Cells were stored incorrectly Obtain new stock and store in liquid nitrogen. Keep the cells in
stock liquid nitrogen until thawing.
Home made freezer stock is not viable Freeze cells at a density recommended by the supplier.
Use low-passage cells to make your own freezer stocks.
Follow procedures for freezing cells exactly as recommeded by
the supplier. Note that the freezing procedure recommended in
this handbook is a general procedure provided as a guideline
only.
Obtain new stock.
Cells were thawed incorrectly Follow procedures for thawing cells exactly as recommeded by
the supplier. Note that the thawing procedure recommended in
this handbook is a general procedure provided as a guideline
only.
Make sure that you thaw the frozen cells quickly, but dilute them
slowly using pre-warmed growth medium before plating.
Thawing medium is not correct Use the medium recommended by the supplier. Make sure the
medium is pre-warmed.
Cells are too dilute Plate thawed cells at high density as recommended by the
supplier to optimize recovery.
Cells not handled gently Freezing and thawing procedures are stressful to most cells. Do
not vortex, bang the flasks to dislodge the cells (except when
culturing insect cells), or centrifuge the cells at high speeds.
Glycerol used in the freezing medium was If stored in light, glycerol is gets converted to acrolein, which toxic
stored in light (if applicable) to cells. Obtain new stock.
Cells grow slowly Growth medium is not correct Use pre-warmed growth medium as recommended by the
supplier.
Serum in the growth medium is of poor Use serum from a different lot.
quality
Cells have been passaged too many times Use healthy, low passage-number cells.
Cells were allowed to grow beyond Passage mammalian cells when they are in the log-phase before
confluency they reach confluence.
Culture is contaminated with mycoplasma Discard cells, media, and reagents. Obtain new stock of cells, and
use them with fresh media and reagents.

Part 7. Appendix
Cell Culture and Transfection Products

Life Technologies™ offers a variety of primary cultures and established cell lines, as
well as reagents, media, sera, and growth factors for your cell culture experiments.
The tables below contain lists of the more commonly used cell lines and other cell
culture products available from Life Technologies™. For more information on Life
Technologies™ and Gibco® products, refer to www.lifetechnologies.com/cellculture.
Cell lines In addition to the mammalian and insect cell lines listed below, Life Technologies™
offers primary mammalian cells and complete cell culture systems, including
keratinocyte, fibroblast, melanocyte, hepatocyte, corneal and mammary epithelial,
large vessel and microvascular endothelial, smooth muscle, and neuronal cell culture
systems. For a comprehensive list of cells, technical resources and related technologies,
visit www.lifetechnologies.com/cellculture.
Product Quantity Cat. no.

Mammalian Cell Lines
293-F Cells, SFM adapted (7.5 × 106 cells) 1.5 mL 11625-019
6
293-H Cells, SFM adapted (7.5 × 10 cells) 1.5 mL 11631-017
6
293FT Cell Line 3 × 10 cells R700-07
6
293A Cell Line 3 × 10 cells R705-07
™
GripTite 293 MSR Cell line 1 kit R795-07
CHO DG44 Cells (cGMP banked) and Media Kit 1 kit A11000-01
Insect Cell Lines

Sf9 Cells (SFM adapted) (1.5 × 107 cells) 1.5 mL 11496-015
Sf21 Cells (SFM adapted) (1.5 × 107 cells) 1.5 mL 11497-013
Mimic™ Sf9 Insect Cells (1 × 107 cells) 1 mL 12552-014
Sf9 Cells adapted in Sf-900™ III SFM (1.5 × 107 cells) 1 vial 12659-017
Sf21 Cells adapted in Sf-900™ III SFM (1.5 × 107 cells) 1 vial 12682-019
Sf9 Frozen Cells (Grace’s media) (1 × 107 cells) 1 mL B825-01
Sf21 Frozen Cells (Grace’s media) (1 × 107 cells) 1 mL B821-01

™ ® 6
High Five Cells, adapted to Express Five SFM (3 × 10 cells) 1 mL B855-02
97
Part 7. Appendix
Media for mammalian

cell culture Life Technologies™ provides you with all of your cell culture needs through its Gibco®
Cell Culture Media, and offers products to support the growth of a range of mammalian
cell lines. All cell culture media products available from Life Technologies™ are tested
for contamination, and guaranteed for their quality, safety, consistency, and regulatory
compliance. In addition to the media listed below, Life Technologies™ offers a large
selection of serum-free and specilized media for culturing primary cells, established
cell lines, and stem cells, as well as for virus production, protein expression, stem
cell differentiation, and cytogenetics. For more information and a complete list of cell
culture media, visit www.lifetechnologies.com/cellculture.
Product* Quantity† Cat. no.

D-MEM (1X), liquid (high glucose with no glutamine) 10 × 500 mL 35053-036
D-MEM (1X), liquid (low glucose with no glutamine) 500 mL 11054-020
D-MEM (1X), liquid (high glucose with GlutaMAX™-I) 10 × 500 mL 10564-029
D-MEM (1X), liquid (low glucose with GlutaMAX™-I) 10 × 500 mL 10567-022
Advanced D-MEM (1X), liquid (high glucose with no glutamine) 10 × 500 mL 12491-023
D-MEM/F-12, liquid, 1:1 (with GlutaMAX™-I) 10 × 500 mL 10565-042
D-MEM/F-12, liquid, 1:1 (with L-glutamine) 10 × 500 mL 11320-082
Advanced D-MEM/F-12, liquid, 1:1 (with no glutamine) 10 × 500 mL 12634-028
Minimum Essential Medium (MEM) (1X), liquid (with no 10 × 500 mL 11090-099

glutamine)
Minimum Essential Medium (MEM) (1X), liquid (with 10 × 500 mL 41090-101

GlutaMAX™-I)
Advanced MEM (Minimum Essential Medium) (1X), liquid (with 10 × 500 mL 12492-021
no glutamine)
RPMI Medium 1640 (1X), liquid (with no glutamine) 1000 mL 21870-084

™
RPMI Medium 1640 (1X), liquid (with GlutaMAX -I) 10 × 500 mL 61870-127
Advanced RPMI Medium 1640 (1X), liquid (with no glutamine) 10 × 500 mL 12633-020
293 SFM II, liquid 1,000 mL 11686-029
CD 293 Medium, liquid 1,000 mL 11913-019
Gibco® Freestyle™ 293 Expression Medium 1,000 mL 12338-018
CD CHO Medium (1X), liquid 1,000 mL 10743-029
CHO-S-SFM II 1,000 mL 12052-098
Gibco® Freestyle™ CHO Expression Medium 1,000 mL 12651-014

™
CD OptiCHO Medium (1X), liquid 1,000 mL 12681-011
™
Recovery Cell Culture Freezing Medium, liquid 50 mL 12648-010
®
Synth-a-Freeze Cryopreservation Medium 50 mL R-005-50
*Most of the media listed in this table are available with L-glutamine, GlutaMAX -I, or no glutamine,
™
with or without phenol red, as well as in powder and liquid formulations. †Also available in different
quantities and packaging sizes.

Part 7. Appendix
Media for insect cell

culture Insect cell culture is a common choice for heterologous protein expression. For large
scale production or basic research, insect cells are able to express large quantities of
protein with complex post-translational modifications. Gibco® insect media from Life
Technologies™ have been formulated for maximum growth and protein yields. For more
information, visit www.lifetechnologies.com.

Grace’s Insect Cell Medium, Unsupplemented (1X), liquid 500 mL 11595-030
Grace’s Insect Cell Medium, Supplemented (1X), liquid 500 mL 11605-094
Sf-900 II SFM (1X), liquid 500 mL 10902-096

™
Sf-900 III SFM (1X), liquid 500 mL 12658-019
Schneider’s Drosophila Medium (1X), liquid 10 × 500 mL 11720-067
IPL-41 Insect Medium (1X), liquid 1,000 mL 11405-081

®
Express Five SFM (1X), liquid 1,000 mL 10486-025
*Most of the media listed in this table are available in powder and liquid formulations. †Also available
in different quantities and packaging sizes.
Serum products for cell

culture Life Technologies™ supplies a wide range of Gibco® animal sera, both bovine and
nonbovine, for cell culture applications, the most widely used being fetal bovine serum
(FBS). The table below lists a small selection of sera available from Life Technologies™.
For a complete list and more information on the use, sources, traceability, collection,
and bottling of serum, refer to www.lifetechnologies.com.

Fetal Bovine Serum (FBS), Certified, Heat-Inactivated 500 mL 10082-147
Fetal Bovine Serum (FBS), Qualified, Heat-Inactivated 500 mL 10100-147
Fetal Bovine Serum (FBS), Certified 1,000 mL 16000-069
Fetal Bovine Serum (FBS), Qualified 1,000 mL 10099-158
OneShot™ Fetal Bovine Serum, Certified 50 mL 16000-077
OneShot™ Fetal Bovine Serum, Qualified 50 mL 26140-111
Bovine Serum 500 mL 16170-078
Bovine Serum, Heat-Inactivated 500 mL 26170-043
Horse Serum 500 mL 16050-122
Horse Serum, Heat-Inactivated 500 mL 26050-088
Porcine Serum 500 mL 26250-084
Goat Serum 500 mL 16210-072
Rabbit Serum 500 mL 16120-099

™
*To ensure supply, Life Technologies sources FBS from the United States, New Zealand, Australia,
and other countries meeting USDA importation requirements (i.e., USDA-Approved). All other serum
products are sourced from New Zealand with the exception of rabbit serum, which is sourced from the
United States. †Also available in different quantities and packaging sizes.
99
Part 7. Appendix
Laboratory reagents for

cell culture The table below lists a small selection of laboratory reagents for cell culture that are
available from Life Technologies™. For more information and a complete list, refer to
Product Quantity* Cat. no.

Balanced Salt Solutions: D-PBS†, EBSS, HBSS‡, PBS
Dulbecco’s Phosphate Buffered Saline (D-PBS) (1X), liquid 1,000 mL 14040-117
Earle’s Balanced Salt Solution (EBSS) (1X), liquid 500 mL 14155-063
Hank’s Balanced Salt Solution (HBSS) (1X), liquid 1,000 mL 14025-076
Phosphate-Buffered Saline (PBS) pH 7.4 (1X), liquid 500 mL 10010-023
Phosphate-Buffered Saline (PBS) pH 7.2 (1X), liquid 500 mL 70013-032
Buffers and Chemicals

HEPES Buffer Solution (1M) 20 × 100 mL 15630-130
Sodium Bicarbonate Solution, 7.5% (w/v) 100 mL 25080-094
Cell Dissociation Reagents

TrypLE™ Express Dissociation Reagent with Phenol Red 500 mL 12605-028
™
TrypLE Express Dissociation Reagent without Phenol Red 100 mL 12604-013
™
TrypLE Select Dissociation Reagent 500 mL 12563-029
Trypsin, 0.5% (10X), liquid, with EDTA 4Na, without Phenol Red 100 mL 15400-054
Trypsin, 0.25% (10X), liquid, without EDTA, with Phenol Red 500 mL 15050-057
Collagenase Type I 1g 17100-017
Collagenase Type II 1g 17101-015
Dispase 5g 17105-041
Trypsin Inhibitor, soybean 1g 17075-029
Supplements
L-Glutamine†, 200 mM (100X), liquid 100 mL 25030-081
GlutaMAX™-I Supplement 100 mL 35050-061
D-Glucose (Dextrose) 1 kg 15023-021
Pluronic® F-68, 10% (100X) 100 mL 24040-032
MEM Amino Acids Solution (50X), liquid 100 mL 11130-051
MEM Non-Essential Amino Acids Solution 10 mM (100X), liquid 100 mL 11140-050
MEM Sodium Pyruvate Solution 10 mM (100X), liquid 100 mL 11360-070
MEM Vitamin Solution (100X), liquid 100 mL 11120-052
2-Mercaptoethanol (1,000X), liquid 50 mL 21985-023

™
CHO CD EfficientFeed Kit 1 kit A10241-01
™
FoamAway Irradiated AOF Anti-Foaming Agent 500 mL A1036902
*Products are also available in different quantities and packaging sizes. †Product is available in liquid
or powder formats. ‡HBSS is available with or without magnesium and calcium, and with or without
phenol red.

Part 7. Appendix
Antibiotics and
antimycotics Antibiotics are used to protect the integrity of your cell culture as well for selection and
establishmeny of cell lines; Life Technologies™ offers a wide selection of antibiotics,
antimycotics and detection kits. For more information, refer to www.lifetechnologies.com.

Antibiotics and Antimycotics
Antibiotic-Antimycotic (100X), liquid 100 mL 15240-062
®
Fungizone Antimycotic, liquid 20 mL 15290-018
Gentamycin Reagent Solution (10 mg/mL), liquid 10 mL 15710-064
Gentamycin Reagent Solution (50 mg/mL), liquid 10 mL 15750-060
Gentamycin/Amphotericin Solution 10 × 1 mL R-015-10
Neomycin Sulfate, powder 100 g 21810-031
Penicillin-Streptomycin, liquid 100 mL 15140-122
Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture 100 mL 15640-055
Selection Antibiotics
Geneticin® Selective Antibiotic, liquid 20 mL 10131-027
®
Geneticin Selective Antibiotic, powder 1g 11811-023
Hygromycin B 20 mL 10687-010
Puromycin Dihydrochloride, Selection Antibiotic, liquid 10 × 1 mL A11138-03
Blasticidin S HCl, Selection Antibiotic, liquid 20 mL A11139-02
Blasticidin S HCl, powder 50 mg R210-01

™
Zeocin Selection Reagent, powder 1g R250-01
Contamination Detection Kits

Cell Culture Contamination Detection Kit - 200 assays 1 kit C7028
MycoFluor™ Mycoplasma Detection Kit 1 kit M7006
*Products are also available in different quantities and packaging sizes.
Growth factors and

purified proteins Life Technologies™ offers an array of highly-potent and highly-pure growth factors,
chemokines, cytokines, and other proteins and protein inhibitors validated for use in cell
culture. These products have been validated in live cell bioassays using Gibco® media. For
more information and a complete list, refer to www.lifetechnologies.com/cellculture.
101
Part 7. Appendix
Accessory products for

cell culture The table below lists a small selection of accessory products for cell culture that are
available from Life Technologies™. For more information and a complete list, refer to

Attachment Factors and Matrices
Geltrex® Reduced Growth Factor Basement Membrane Matrix 5 mL 12760-021
™
AlgiMatrix 3D culture System 6-well plates 1 plate A10982-01
™
AlgiMatrix 3D culture System 24-well plates 1 plate 12684-023
™
AlgiMatrix 3D culture System 96-well plates 1 plate 12684-015
Human Plasma Fibronectin 5 mg 33016-015
Natural Mouse Laminin 1 mg 23017-015
Instruments
Countess® II Automated Cell Counter 1 unit AMQAX1000
®
Countess II FL Automated Cell Counter 1 unit AMQAF1000
Mallassez Hemocytometer 1 unit 99503

®
Qubit 3.0 Fluorometer 1 unit Q33217
*Some of the products listed in the table are also available in different quantities and packaging sizes.
Transfection reagents Life Technologies™ offers the most complete collection of cationic lipid-based
transfection reagents with exceptional performance that can be used for delivery of
DNA, siRNA, oligonucleotide, and RNA. The table below lists a small selection of
cationic-lipid transfection reagents that are available from Life Technologies™. For more
information and a complete list, refer to www.lifetechnologies.com/transfection.

Lipofectamine® 3000 Transfection Reagent 1.5 mL L3000-015
Lipofectamine® 2000 Transfection Reagent 1.5 mL 11668-019
Lipofectamine® 2000 CD (Chemically Defined) Transfection 1 mL 12566-014

Reagent
Lipofectamine® RNAiMAX Transfection Reagent 1.5 mL 13778-150

® ™
Lipofectamine LTX with Plus Reagent 1 mL 15338-100
®
Lipofectamine MessengerMAX 1.5 mL LMRNA015
®
Invivofectamine 2.0 Reagent 1 mL 13775-01
™
ExpiFectamine 293 Transfection Kit 1 × 1 L culture A14524
™
FreeStyle MAX Reagent 1 mL 16447-100
®
Cellfectin II Reagent 1 mL 10362-100
*Products are also available in different quantities and packaging sizes.

Part 7. Appendix
Neon® Transfection
System The Neon® Transfection System efficiently delivers nucleic acids, proteins, and siRNA
into all mammalian cell types, including primary and immortalized hematopoietic cells,
stem cells, and primary cells, with a high cell survival rate. For more information on
the Neon® Transfection System and optimized Neon® transfection protocols for many
commonly used cell types, refer to www.lifetechnologies.com/neon.
Product Quantity Cat. no.

®
Neon Transfection System 100 µL Kit 192 reactions MPK10096
®
Neon Transfection System 10 µL Kit 192 reactions MPK1096
Neon® Transfection System 1 each MPK5000
Neon® Transfection System Starter Pack 1 pack MPK5000S
Neon® Transfection System Pipette 1 each MPP100
Neon® Transfection System Pipette Station 1 each MPS100
Neon® Transfection Tubes 1 pack MPT100
RNA interference RNAi is a specific, potent, and highly successful approach for loss-of-function studies
in virtually all eukaryotic organisms. Life Technologies™ has developed two types of
small RNA molecules that function in RNAi, short interfering RNA (siRNA) molecules
and microRNAs (miRNA), and offers a variety of products for RNAi analysis in vitro
and in vivo, including libraries for high-throughput applications. Your choice of tool
depends on your model system, the length of time you require knockdown, and other
experimental parameters.
In addition, Life Technologies™ offers the most complete collection of cationic lipid-
based transfection reagents with exceptional performance that can be used for delivery
of assorted RNAi reagents, including shRNA and miR RNAi vectors and synthetic
molecules such as siRNA, Stealth RNAi™ siRNA, and Dicer-generated siRNAi pools.
Further, cell specific RNAi transfection protocols have been developed using these
transfection reagents for many popular cell lines.
For more information and a complete list of RNAi products available from Life
Technologies™, refer to www.lifetechnologies.com/rnai.
103
Part 7. Appendix
Additional Resources
Mammalian and insect
cell cultures For more information on mammalian and insect cell culture, cell type specific protocols,
and additional cell culture products, refer to Mammalian Cell Culture and Insect Cell
Culture portals on our website.
Cell and tissue analysis Understanding the structural and functional relationships of cells and tissues is critical
to advancements in key research disciplines, including molecular biology, genetics,
reproductive function, immunology, cancer and neurobiology. Key components of cell
and tissue analysis are cell viability and proliferation, cell signaling pathways, cell cycle
analysis, and cell structure. Life Technologies™ has a broad portfolio of reagents and
kits for cell and tissue analysis, including Molecular Probes® Fluorescence Products and
technologies as well as Dynal® Bead-based Solutions for cell isolation and expansion.
From antibodies and stem cell research products to benchtop instruments like the
Countess® II Automated Cell Counter and the Qubit® Fluorometer, Life Technologies™
has the tools essential for cellular analysis research. For more information, refer to the
Cell & Tissue Analysis portal on our website.
Safety data sheets Safety Data Sheets (SDS) are available at www.lifetechnologies.com/sds.
Certificate of analysis The Certificate of Analysis provides detailed quality control and product qualification
information for each product. Certificates of Analysis are available on our website. Go
to www.lifetechnologies.com/support and search for the Certificate of Analysis by
product lot number, which is printed on the box.
Technical support For more information or technical assistance, call, write, fax, or email our award
winning Technical Support.
Limited product warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set
forth in the Life Technologies’ General Terms and Conditions of Sale found on Life
Technologies’ website at www.lifetechnologies.com/termsandconditions. If you have
any questions, please contact Life Technologies at www.lifetechnologies.com/support.

Part 7. Appendix
References
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Anson, D. S. (2004) The use of retroviral vectors for gene therapy-what are the risks?
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107
Part 7. Appendix
Notes

Part 7. Appendix
Notes
109
Part 7. Appendix
Notes

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Cell Culture Basics Handbook

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Purpose of the Handbook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Introduction to Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2. Cell Culture Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Cell Culture Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Cell Culture Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Cell Culture Basics | iii

Aseptic Technique Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

3. Cell Culture Basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

4. Cell Culture Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Guidelines for Maintaining Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

iv | Cell Culture Basics

Dissociating adherent cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Subculturing Adherent Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Subculturing Suspension Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Thawing Frozen Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Gene Delivery Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Calcium phosphate co‑precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Cationic Lipid-Mediated Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Virus-Mediated Gene Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Neon® Transfection System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Selection of Stable Transfectants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Reporter Gene Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

RNAi and Non-coding RNA Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Factors Influencing Transfection Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

vi | Cell Culture Basics

Type of molecule transfected . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Selecting a Transfection Method (non-viral) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Selecting a Viral DNA Delivery System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Guidelines for Plasmid DNA Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Optimization of Plasmid DNA Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Selection of Stable Transfectants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Selecting a RNAi Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Guidelines for RNA Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Optimization of siRNA Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

Cell Culture and Transfection Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Additional Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

viii | Cell Culture Basics

Purpose of the Handbook

Cell Culture Basics | 1

Introduction to Cell Culture

Finite vs. continuous

2 | Cell Culture Basics

• Fibroblastic (or fibroblast-like) cells are bipolar or multipolar, have elongated

• Lymphoblast-like cells are spherical in shape and usually grown in suspension

The fundamental objective of any biosafety program is to reduce or eliminate exposure

Biosafety Level 1 (BSL-1)

Biosafety Level 2 (BSL-2)

Biosafety Level 3 (BSL-3)

Biosafety Level 4 (BSL-4)

4 | Cell Culture Basics

SDSs for all Life Technologies products are available at www.lifetechnologies.com/sds.

• Always wear appropriate personal protective equipment. Change gloves when

Cell Culture Equipment

Expanded equipment • Aspiration pump (peristaltic or vacuum)

6 | Cell Culture Basics

Cell Culture Laboratory