Basics of Cell

Culture
A student laboratory manual

By Golnar Afshar PhD

Table of Contents:

Introduction 3 Laboratory Exercises

Laboratory Safety 3 Aseptic Technique Exercise30 30
Laboratory Notebooks5 5 Plating Cells from Frozen 31
Stocks
Cell culture Laboratory 7 Count and 33
Equipment Plate 104 Cells in a T25
Laminar-Flow Hood 7 Survival Assay- 35
Sensitivity to UV Exposure35
Inverted Microscopes 8 Live and Dead Cell 41
Identification by Using
Propidium Iodide and Calcein
AM41
Fluorescent 9 Transfection 44
Inverted Microscopes
Clinical Centrifuge 10 Freezing Cells 48
Incubator 11 Stem Cell Differentiation 50
370C Water Bath 12 Appendix A- Serial Dilution 54
Refrigerator and Freezers 12 Appendix B- Practice problems 55
Biohazard Waste Containers 12 Glossary 57
Cell Culture Vessels 12 References 60
Pipettes 13

Contamination 15 Edited by: Deborah Blancas, Carin

Zimmerman Ph.D., and Edie Kaeuper
Common Laboratory Procedures 19 Ph.D.
Media Preparation 20

Routine Cell Culture 23 Note: Terms bolded in the paragraphs of

Maintenance this text can be found in the glossary.
(Feeding and Subculturing)
Counting Cells Using a 26
Hemocytometer

2
Basics of Cell Culture The class meets once a week for nine weeks.
Each class lasts four hours with a brief
Cell culture--a method of multiplying cells lecture, followed by a laboratory exercise.
under controlled, laboratory conditions--is
used by scientists to study cellular functions, Laboratory safety and professional
structures and behaviors. Cell culture is conduct
rooted in tissue-culturing techniques first
developed in the early twentieth century. Safety policies are essential for this class
Initially, tissue cultures were made by using and must be followed not only for your own
fragments of tissue or organs called protection but for the protection of others as
explants. However, this form of culture well. Failure to follow these procedures will
yielded tissue with very limited growth not only severely affect your grade, but may
capabilities. also lead to health complications. The
following policies must be adhered to while
One of the first significant advances in in the laboratory.
tissue growth was the ability to isolate
individual cells from tissue for use in the 1. Eating, drinking or chewing gum is
laboratory. But, these individual cells were prohibited.
also limited by a finite number of divisions,
a phenomenon known as Hayflick’s Limit 2. Keep your hands away from your mouth
(Hayflick and Moorhead, 1961). This during the laboratory class.
limitation was overcome by the discovery in
the 1950s of special cervical carcinoma 3. Horseplay or running in the laboratory is
cells, known as HeLa cells, which have an not allowed.
indefinite lifespan (Gey et al. 1952). Due to
modern advances, cells can now be easily 4. You must wear a laboratory coat during
grown in large numbers and stored the laboratory session.
indefinitely.
5. You must disinfect your laboratory work
In addition to studying cellular structure and area prior to and after each class.
functions, cell cultures are used for a variety
of applications, including toxicology studies, 6. You must wash your hands before starting
drug development, recombinant protein your work with the cells and after
production, diagnosis, treatment and more. completion of the laboratory class. Gloves
are required and will be provided for
This course is designed to expose students to procedures that use potentially hazardous
the cell culture laboratory environment and material.
to introduce the fundamental concepts and
techniques of mammalian cell culture. 7. Laboratory accidents, spills and injuries
must immediately be brought to the attention

3
of the instructor.

8. Friends or visitors are not permitted in the

laboratory.

9. Closed-toe shoes and long pants must be

worn.

10. Biohazard material and contaminated

cultures must be disposed of according to
the instructions.

11. Dispose of the glass Pasteur pipettes and

micropipette tips in the biohazard sharp
pouches. Dispose of serological pipettes into
the dedicated disposal trays beside your
work area; the pipettes will be sterilized
before disposal.

12. Aspirate liquid waste into waste bottles,

before throwing flasks and plates into trash.

13. Cell phones must be turned off for the

laboratory period.

14. Familiarize yourself with the locations

of emergency safety equipment in the
laboratory, such as first aid kit, eye-wash
station, and fire extinguisher.

4
Laboratory Notebooks 2. All entries must be made in black or blue
ink, except drawings or sketches, which may
be in pencil.
Accurate record keeping is essential to the
progress of science. Scientists place great
3. You must start your notebook with a table
importance on recording the procedures and
of contents that lists titles of lab exercises
data during the experiment. The original
and their corresponding page numbers.
hand-written notes and records are of great
importance for accuracy since rewriting
4. Every page should be numbered
notes or relying on memory can increase the
sequentially with no pages missing or torn
risk of introducing errors to the records.
out.
Laboratory notes need to be accurate,
readable and organized so that another
5. If you are repeating a protocol or part of
scientist, who is unfamiliar with the
an experiment, it is acceptable to write "as
experiment, can follow the procedures and
done previously on page A" without
make sense of the data.
rewriting the entire entry.
It is very important that the mistakes made
6. Do not leave any pages blank. Cross out
during a procedure or changes made to the
blank spaces in your notebook. This will
protocol are recorded in the notes. Mistakes
prevent adding notes to previously recorded
often lead to new questions and discoveries;
data.
and knowing that a procedure was changed
due to mistakes or unexpected
7. Entries must be made in a chronological
circumstances help to interpret the data.
order. If an experiment runs over for several
days, you can write at the bottom of your
Laboratory notebooks are often used as legal
entry "continued on page B". When you
records for patenting discoveries and for
continue an entry on another page start with
verification of compliance with regulations.
"continued from page C". Remember to note
Therefore, it is important that the records are
the page numbers in your table of contents.
properly documented in a way that makes it
difficult to change, add or delete the data
8. Figures and graphs must have a title and
that has been recorded. In some industries
be clearly labeled.
and research institutions the notebooks are
signed by the supervisors and kept in a safe.
9. Graphs or material written on a separate
piece of paper must be glued to your
Rules for laboratory notebooks:
notebook. Do not use staples or tape.
Student notebooks will be collected at the
10. Do not use white-out. If you make a
end of the semester and graded, based on
mistake, just cross the incorrect words with
organization and completeness of the notes.
a single line so that it is still readable.
1. All laboratory notebooks must be
11. Leave a space at the bottom of every
permanently bound. Binders and spiral-
page for the student and the instructor to
bound notebooks are unacceptable. This will
sign and date.
prevent the addition and/or removal of
pages.

5
12. The date must be in the international
scientific format, “day/month/year”.

13. Your name must be on the cover of your

notebook and on the three side edges.

14. If you follow the steps outlined above,

you will have an entry detailing the lab work
you performed on that day.

15. Each entry (Fig. 1) should include:

-Title
-Purpose or Objectives (may be kept simple
1-2 sentences)
-Materials (a list of items that you need to
perform the experiment.)
-Methods or Protocol- (step by step
procedure. Can be in form of diagrams.) The
protocols provided in this manual are
general procedures that may have to be
altered for your specific cell line or purpose.
Use your own words to simplify the steps in
a way that is easy to follow.
- Observations of the status of your cells Figure 1- An example of a laboratory
-Data and Results notebook entry.
-Answers to analysis questions which forms
your discussion and a conclusion

6
Cell Culture Laboratory The following guidelines must be followed
Equipment while working inside the hood:

Equipment commonly used in a cell-culture 1. Make sure that the UV-germicidal lamp is
laboratory: turned off.

Laminar-flow hood 2. Open the glass shield to the allowable

level and switch on the blower. Wait about
Most cell culture procedures are performed 10 minutes before starting work to allow for
inside laminar-flow hoods (Fig.2). Laminar- the air inside the cabinet to be filtered.
flow hoods, or biological safety cabinets,
provide a clean working environment to 3. Wipe down the inside surface of the hood
prevent contamination of cell cultures. The with alcohol before starting work.
air is filtered and cleaned of particles before
blown into the cabinet. Additionally, the 4. All equipment and supplies used inside
flow of air in the hood is in smooth parallel the hood must be clean and wiped with
lines which creates a “curtain” to separate alcohol.
inside from outside. Some laminar hoods
are equipped with a UV-germicidal lamp to 5. Do not overcrowd the hood. Keep your
sterilize the contents inside while not in use. space clean and organized.
The UV lamp must be turned off before
working in the hood to prevent exposure to 6. Wipe any spills immediately with alcohol.
hazardous UV light.
7. Do not block the air filters and blowers.

8. Try to work towards the middle of the

hood while being careful not to block the
front area where the air filter is located.

9. Do not make rapid movements since it

will disrupt the laminar flow of the air.

10. Clean the inside of the hood and wipe

down with alcohol after your work is
finished.

11. Turn off the blower and close the glass

shield before you leave.

Figure 2- A laminar flow hood.

7
Inverted microscopes above the stage concentrates and focuses the
light from the light source.
Inverted microscopes are used to observe the
cells in culture (Fig. 3). Inverted When observing your cells:
microscopes are a type of microscope with
1. Wipe the stage with alcohol.
the objective lenses below the stage and the
light source and the condenser above the 2. If you need to clean the lenses, only use
stage. These microscopes are especially lens paper to avoid scratching any of the
suitable for observing cells that are attached microscope’s lens.
to the bottom of the plates and/or flasks.
3. Place your plate or flask of cells on the
stage.

4. Turn on the light.

5. Turn the objective lens turret to the

smallest magnification (4X).

6. Look at the cells by using the ocular

lenses while moving the flask slightly and
focusing to find the cells.

7. Turn the turret to a higher magnification.

Figure 3- An inverted microscope. Usually 10X is sufficient for routine
observations.
The flask/plate is placed on the stage and the
image is focused by turning the focus knobs. 8. Adjust the focus knobs for the best
The objective lenses under the stage can possible image. To focus for both of your
provide 4X, 10X, 20X and sometimes more eyes, close your right eye and focus with the
magnification of the image. The objective focus knob first. Then close your left eye
turret, which holds the objective lenses, can and focus by turning the right ocular lens.
be turned to place the appropriate objective
lens in place. The phase rings above the 9. You may want to slide a phase ring in the
stage are placed in the light path to change light path for a clearer image. For observing
the phase of the light when going through some cell types, phase rings may be helpful.
different structures of the cells in order to
make the transparent structures more visible 10. Turn off the microscope after you are
to the eyes. Different phase rings are located finished.
on a slider and can be placed in the light
path for a clearer image. The condenser

8
Fluorescent inverted microscopes completely done with your work. You
cannot turn the light back on again until it
Fluorescent microscopes are inverted has cooled off completely, which may take
microscopes, used to observe cells and up to 1-2 hours.
molecules that have been labeled with
fluorophores. Fluorophores are molecules 3. Block the light path to prevent your cells
that can absorb energy of light at specific from overexposure to the high intensity
wavelengths and emit less energetic light. Fluorophores that are exposed to
fluorescent light. Fluorescent microscopes continuous light will eventually lose their
are equipped with filters that will separate fluorescent properties, a phenomenon called
the absorbed light from the emitted “photobleaching”.
fluorescent light. A set of filters are mounted
on a block called the filter cube. 4. Place your plate or flask of cells on the
stage and turn on the regular microscope
Fluorescent microscopes usually have light.
several filter cubes with different sets of
filters appropriate for observing 5. Using the lower magnification objective
fluorophores that emit light at different lens, find your specimen and focus.
wavelengths. The filter cubes are
conveniently located on a turret that can be 6. Turn to the appropriate higher
rotated in order to place the appropriate cube magnification objective lens and adjust the
in place to observe a specific fluorophore. focus.
Use of fluorophores and fluorescent
microscopes has enabled scientists to view 7. Turn off the regular microscope light and
cellular structures and to study molecular unblock the high-intensity light path.
functions and interactions.
8. Place the appropriate filter cube in place.
Your instructor will demonstrate the proper
use and care of the fluorescent microscopes 9. Observe the cells and make notes.
in detail. The following are some general
rules to be followed when using a 10. Remember to provide protection against
fluorescent microscope: photobleaching by blocking the light path
when not observing the cells.
1. For better view, turn off the lights in the
room. 11. Turn off the light source when your
work is finished.
2. Turn on the high-intensity light source
(usually a xenon-arc or a mercury-vapor 12. Do not put the plastic cover on the
lamp) and allow about 10 minutes for warm microscope until the light source has cooled
up. Do not turn off the lamp until you are down.

9
Clinical Centrifuge that are properly balanced, i.e. weigh the
same amount.
Clinical centrifuges (Fig. 4) are used to
concentrate the cells and to separate the cells 3. Sometimes you must prepare a separate
from the media or other reagents. Slow- balancing tube of the same size by filling it
speed clinical centrifuge must be used in with tap water. The balancing tube must be
order to prevent damage to the cells. For of the same weight as the tube that needs to
routine spinning of the cells, speed of 80- be centrifuged.
100g (gravitational force) is sufficient.
Higher speeds may damage the cells. Proper 4. Place the two tubes that have been
care and operation of the centrifuge will be balanced into two opposing slots of the
explained in detail by your instructor. centrifuge.

5. Close the safety lid.

6. Set the centrifuge to the appropriate speed

and time, then turn it on.

7. Stay close to the centrifuge for the first

minute to make sure the centrifuge is
running smoothly. If the centrifuge is not
balanced properly, it will vibrate. Some
centrifuges will turn off automatically if
unbalanced. You need to turn off the older
model centrifuges manually as soon as you
sense the imbalance and vibrations.
Figure 4- Two different models of clinical Continuation of the spin while the centrifuge
centrifuges. is imbalanced will damage the centrifuge.

General rules for using a centrifuge:

8. Do not open the safety lid while the motor
is running.
1. Transfer the liquid suspension to the
appropriately sized centrifuge tubes. Not all
9. Wipe any spills that might have occurred
tubes will be able to survive the forces of the
after centrifugation.
centrifugation, so please use the tubes
specifically manufactured for centrifuge you
are using.

2. Weigh your tubes with their contents on a

pan-balance to make sure that the tubes are
of equal weight. Fig. 5 depicted two tubes

10
 are connected to a CO2 gas tank. CO2 gas is
injected inside the incubator and distributed
by a fan or natural convection. CO2 levels
are usually maintained at 5%. CO2 interacts
with the bicarbonate buffer in the cell
culture medium. This interaction stabilizes
the pH at about 7.4. Uncorrected changes in
medium pH can damage the cells.

H2O + CO2H2CO3  H+ + HCO3-

Figure 5- Use a scale to balance your tubes It is important to periodically check the tank
for centrifugation. gauges and take care that CO2 levels are
maintained in your incubator. Keep the
Incubator incubator closed at all times and avoid
frequent opening of the door to prevent loss
The incubators (Fig. 6) provide the of the CO2 gas.
appropriate environment for the cells to
grow. Cell culture incubators have three
main functions:

1. Constant temperature- Incubators can be

set to a specific temperature appropriate for
the cells. For mammalian cells the
temperature is kept at 37oC, which is the
optimal temperature for their growth.

2. Humidity- Although cells are kept in

liquid media, smaller dishes that hold less
liquid require a humid environment to
prevent evaporation of the media. Usually a
container filled with sterile, distilled water is Figure 6- A cell culture incubator connected
placed in the incubator to provide humidity. to the CO2 tank.
The water needs to be replaced with fresh
Some incubators are also able to control the
sterile distilled water regularly to prevent amount of oxygen available to the cells.
growth of microorganisms and reduce the
possibility of contamination. The optimal temperature and humidity
provides an environment suitable for the
3. CO2 gas is needed to keep the pH growth of bacteria and other
balanced; that is why cell culture incubators microorganisms. Therefore, it is necessary

11
to clean the incubators frequently to prevent Biohazard waste containers
growth and spread of contamination.
Potentially hazardous material must be
0
37 C water bath disposed of properly in biohazard waste
containers and sterilized before disposal.
The media and most of the solutions used Biohazard material must be handled
for cell cultures are kept in the 4oC according to federal, local and institutional
refrigerator. The cells, however, are kept in regulations. Your instructor will explain the
the 37oC incubator. In order to prevent proper disposal of material in class. It is
shocking the cells with cold temperature, the very important to comply with the rules to
media and reagents are warmed up in the prevent the spread of potentially hazardous
water bath before use. The warm water in material to the environment.
the water bath is the ideal environment for
the growth of microorganisms and Cell culture vessels
contaminants. Therefore, the water bath
needs to be cleaned and the water replaced Most cells in culture need to attach to a
with fresh distilled water routinely. Bottles substrate in order to divide and grow. These
and containers that have been warmed up in cells often form a monolayer and cover the
the water bath must be wiped down surface available to them. Cells that require
carefully with alcohol before being attachment for growth are said to be
transferred to the hood. anchorage-dependent. Hematopoietic cells
and a few other cell types can grow in liquid
Refrigerator and freezers suspension without attachment. These cells
are said to be anchorage-independent cells.
Most reagents and solutions used for cell The vessels used to grow anchorage-
culture are kept in the refrigerator for short- dependent and anchorage-independent cells
term storage. Some of the reagents can be need to be designed in a way to support the
kept in the -20oC freezer for longer term proper growth of these two cell types.
storage. Cell-culture facilities often have a
-80oC freezer for storage of some of the In most laboratories, disposable polystyrene
reagents and short-term storage of frozen plastic vessels are used to grow anchorage-
cells. dependent cells (Fig. 7). The vessels are flat
at the bottom to provide a surface for cell
For long-term storage, cells are kept in growth. The bottom surface of the culture
liquid nitrogen tanks. The temperature of vessels are coated by molecules such as
liquid nitrogen is -196 oC. Cells can be kept poly-L-lysine, laminin, gelatin, or
frozen in liquid nitrogen for many years. fibronectin. These mimic the natural
The very cold temperature of liquid nitrogen extracellular matrix and allow the cultured
is hazardous; therefore, thick gloves must be cells to attach. There are three types of
worn when opening the tank with great care. commonly used culture vessels used for

12
anchorage-dependent cells: flasks, dishes, Pipettes
and multi-well plates. All three types can be
of different sizes with different surface area. Pipettes are used for transfer of specific
The choice of the vessel depends on the volumes of liquid. Two kinds of pipettes are
nature of the procedures and personal commonly used in laboratories:
preference.
1. Serological pipettes can measure liquids
between 0.1 to 50 mls. You must choose the
appropriate size pipette for the volume that
you are transferring. Serological pipettes
are marked with calibrated lines to allow
measurement of accurate volumes. The
maximum volume that can be transferred
and the size of the pipette’s subdivisions are
Figure 7- Cell printed close to the top of the pipette.
culture dishes and
Serological pipettes require the use of
multi-well plates
(A). Cell culture electrical or manual pumps to draw and
flasks (B) with release liquid.
vented caps (C).
2. Micropipettes are used to transfer small
volumes of liquid, between 1-1000ls
(Fig.8). You must choose the micropipette
The vessels used to grow anchorage- of the appropriate size to use for the volume
independent cells do not need to be treated you are transferring. The range of volumes
for cell attachment. Sterile, stirrer bottles are that can be transferred is written on the
normally used for agitation of the culture micropipette. It is important to stay within
and to keep the cells in suspension. the allowable range. Setting up the pipette to
volumes above or below the allowable range
Both cell types need exchanges of gases (O2 will damage the pipette and will result in
and CO2) for growth. Therefore the cell inaccurate measurements. Micropipettes fit
culture vessels must allow gases to enter. tips that are specific for the specific
Dishes and plates have loose fitting lids and volumes. The tips are kept in color-coded
the caps of the flasks must be closed loosely sterile boxes.
to allow gases to go in. Some flasks have
vented caps with an opening that is covered When using a micropipette:
by a filter to allow gases in, but prevent
entrance of contamination (Fig. 7C). 1. Choose the appropriate size micropipette.

2. Set the dial to the desired volume to be

transferred.

13
3. Open the appropriate size tip box in a
sterile environment (inside the hood).

4. Pick up a tip using the pipette. Do not use

your fingers to fit the tip on the pipette.
Remember to shut the box after the tip is
removed to maintain sterility.

5. Press down gently on the plunger using

your thumb to the first point where you feel
resistance.
6. Place the tip inside the liquid just below
the surface.
Figure 8- Three different sized
7. Gently release the plunger to draw the micropipettes (p20, p100 and p1000) with
liquid in. If the plunger is released too their appropriate tips (A). View of the
micropipette tops (B).
rapidly the liquid will aerate into the
micropipette and will increase the chances
of contamination.

8. To expel the liquid, hold the tip inside the

vessel, touching against the side of the
vessel while holding it slightly tilted. Press
down on the plunger, all the way to the
bottom (past the first resistance point).

9. To dispose of the used pipette tip, hold

the pipette above the disposal bucket and
eject the tip into disposal by pressing the
ejection button.

If used properly, micropipettes can measure

accurate small volumes of liquid. They are
very sensitive and expensive tools and must
be handled with great care.

14
Contamination The warm temperature of the incubators and
the rich media in cell cultures provide an
Cell-culture contamination is a very ideal environment for the growth of various
common problem in all cell-culture microbial contaminants. Some of the most
facilities. Even the most experienced common microbial contaminants in cell
scientist encounters contamination problems cultures are bacteria, fungus, viruses,
once in a while. Contamination can be mycoplasma and other microorganisms.
frustrating and will lead to the loss of the Some microbial contaminations such as
culture, time, effort, and of course is bacteria and fungus are visible and can be
expensive. Although eliminating the detected easily. Bacterial growth in the cell
problem of contamination is not completely culture will often acidify the media. The
possible, it can be very well be managed and phenol red component in the media is a pH
reduced. Careful handling of the cultures marker and will turn yellow at low pH.
and reagent preparation and following good Therefore, a yellow-colored media may be
laboratory practices can reduce the an indication of bacterial contamination. The
contamination problem significantly. bacterial growth at later stages looks cloudy
and can easily be detected with the naked
Cell-culture contaminants can be divided eye. Examining the cells under the
into two major categories; chemical and microscope can also help in detecting
microbial. Chemical contamination occurs bacterial contamination (Fig. 9). Bacteria
when an unwanted chemical substance is cells are much smaller than mammalian cells
present in the media or any of the reagents. and look like tiny dots that cover the surface
The unwanted chemical may be harmful to of the cells and cover the empty spaces of
the cells or may affect their behavior and, the flask/plate in between the cells. Use of
therefore, affect the results of the antibiotics in the media can prevent growth
experiment. Chemical contamination is of some bacteria. However, routine use of
usually not visible and difficult to detect. antibiotics is not recommended, since it
Common sources of chemical contamination encourages the growth of more aggressive,
are the media components, water and other antibiotic-resistant bacteria.
reagents. To prevent this kind of
contamination, care must be taken when Mold contamination is also easily detected.
measuring and preparing the media and The fuzzy-looking mold growth can be seen
other solutions. Presence of alcohol in cell with the naked eye. Under the microscope
cultures is a common chemical you may be able to see the fungi hyphae
contamination problem, since it is routinely strands and the spores in the cell culture.
used to clean the work environment. When
spraying alcohol you must make sure that Yeast is another form of fungus that may
there are no open cell cultures or reagent contaminate cell cultures. Yeast cells are
bottles/tubes in the area. circular and smaller in size than mammalian

15
cells and can be detected under the line. Proper labeling of the culture dishes
microscope. and paying attention to the labels is critical
and can significantly reduce this problem.

Prevention of contamination and aseptic

techniques

Aseptic techniques are a series of

techniques and practices used to reduce the
chances of contamination of the cultures by
microorganisms and to protect laboratory
workers from contamination by cell cultures
and other potentially hazardous material.
Figure 9- Mammalian (CHOk1) cells
contaminated with yeast. Some of the common sources of cell culture
contamination in a laboratory are non-sterile
solutions and supplies, air-borne dust,
Other types of microbial contamination such
laboratory personnel and unclean
as viruses and mycoplasma, are invisible to
equipment, such as the water bath, incubator
the eyes and are, therefore, more difficult to
and laminar hood. Below is a list of some
detect. Mycoplasma are very small
practices and aseptic techniques that can
microorganisms that can grow rapidly in cell
reduce the chances of contamination:
cultures without being visible. Their
presence affects cellular growth and
1. Maintain and clean the equipment
metabolism. Viruses are very small particles
routinely. Wipe down the laminar hood with
that often infect cells and destroy them.
alcohol before and after every use. The hood
Most laboratories perform routine molecular
should also be cleaned more extensively
tests to detect the presence of mycoplasma
once a month. The incubator shelves need to
and some of the common viruses. Cultures
be cleaned and sterilized routinely and the
that are found contaminated are destroyed
water container in the incubator must be
immediately.
replaced every week. The 37oC water bath
has to be cleaned once a week. All other
Another form of cell culture contamination
equipment must also be cleaned routinely. In
is cross-contamination by a different cell
addition, the equipment must be cleaned
line. Scientists often may grow more than
when spills have occurred or contamination
one cell line in the laboratory. If careless, a
had been detected.
worker may mistake one cell line for
another; and two or more cell lines can
2. All equipment and supplies must be
easily become mixed up. This can be a
wiped down with 70% Ethanol, if possible,
serious problem, and will invalidate all of
and be cleaned before being used for cell-
the results obtained from a particular cell

16
culture handling. Remember, wiping down 11. Open the wrapped and sterile serological
with alcohol cleans, but does not sterilize the pipettes inside the hood. You can only touch
equipment. the upper part of the pipette with your
fingers. Be careful not to touch the lower
3. Do not put your notebooks, papers and portion of the pipette to anything that is not
pens inside the hood. Everything in the hood sterile.
must be clean.
12. Use new sterile serological pipettes and
4. Do not cough or sneeze while facing the micropipette tips in between solutions and
hood. cells.

5. All solutions and reagents used for cell 13. Do not open the tops of the culture
culture must be sterile. vessels, the sterile solution containers or the
pipette and tip boxes outside of the laminar
6. To keep from contaminating your cells hood.
wash hands before and after handling your
cells. Also you should wear a clean lab coat. 14. While working, be careful not to touch
A clean lab coat covers your clothes that the rims or the interiors of tubes, flasks and
may be harboring bacteria and other their caps.
microorganisms.
15. Micropipettes can be wiped clean with
7. Always have back up cultures and freeze alcohol before use, but they are not sterile.
your cells frequently. It is wise to freeze Only the tip is sterile, therefore, only the tip
some of your cells when possible, so that in should touch the solutions and the interiors
case of contamination, you have frozen cells of the containers. Be careful if you use a
as back-ups that can be defrosted and used. micropipette inside of a tall tube. It is
preferred to use sterile serological pipettes
8. Aliquot (distribute) solutions and reagents instead, if possible.
into smaller volumes in several sterile
containers, and use one aliquot at a time. 16. Always write a protocol ahead of time
This way, if one aliquot gets contaminated, and get organized before starting work.
you can use the other. Disorganization and confusion increase the
chances of contamination.
9. NEVER use other people’s media and
solutions. This is how contamination is 17. Do not overcrowd your hood and do not
spread among cultures. block the flow of air in the hood.

10. Label your reagents and cultures 18. Always observe your cells before
properly to prevent mix-ups. starting a procedure and destroy

17
contaminated cultures immediately (see 4. Inform other lab personnel who are
below). sharing the incubator, so that they are aware
of the possible contamination.
19. Do not leave the tops of culture vessels
or reagent bottles open when they are not in 5. If the contamination problem is
use. widespread among your cultures, discard all
media and reagents that you have been using
20. Wipe the spills on the hood surface or and start new.
the exteriors of bottles and flasks
immediately with alcohol before it dries out
and you forget.

21. When in doubt, do not use. If you are not

sure if a solution or pipette is sterile or not,
just assume the latter and do not use it for
cell culture. You can put aside the non-
sterile solutions and pipettes to be used for
other procedures in the laboratory.

What to do in case of a microbial

contamination

Contaminated cultures must be destroyed

and disposed of immediately to prevent
further contamination.

1. Add a 10% bleach solution to the

contaminated culture vessel and wait for 10
min. The 10% bleach solution will be clearly
labeled for ease of use.

2. Discard the bleached culture in the sink

with running water. Dispose of the
flask/plate in trash.

3. Clean your work area and equipment that

has been in touch with the contaminated
culture.

18
Common Laboratory Procedures:

Important notes- For all procedures

remember to:
 Practice good aseptic techniques.

 Always wash your hands and clean

your work area before and after a
procedure.

 Always observe your cells before

any procedure, to make sure the cells
are healthy and not contaminated.

 For most routine procedures you

may have to sacrifice extreme
accuracy for the sake of speed. It is
important to work fast when cells are
outside of the incubator and
especially when cells are trypsinized
(enzymatically removed from plates
or flasks) since they are under a lot
of stress and may die. Therefore, it is
important to be organized and do one
procedure with one culture at a time,
if possible.

19
I. Media Preparation
 Amino acids, which are necessary
Cells in culture are missing the tissue and building blocks of proteins.
systemic interactions that normally occur in
the body of an organism to support their  Vitamins, which are needed to promote
growth. Therefore, cell cultures have cell survival and growth.
additional needs to those of cells in a whole
animal. Cells need an abundant source of  Balanced salt solution, which is an
easy to use nutrients to enable them to isotonic mixture of ions to act as
divide rapidly in culture. Formulations for cofactors for enzymatic reactions, cell
cell culture media are designed to mimic adhesion, etc.
natural conditions as much as possible with
additional supplemental nutrients and  Phenol Red dye, which has no
building block components required for nutritional value but is a pH indicator.
cellular growth. Most cells are grown in a The color of phenol red changes from
basal media, containing nutrients, vitamins orange/red at pH 7-7.4 to yellow at
and minerals supplemented with animal acidic lower pH and purple at basic
serum, containing additional nutrients, higher pH environments. The color
growth factors and hormones. This type of change is used to monitor the pH of the
media is said to be “undefined-media” media.
since the exact components of the
supplemental serum are unknown. The  Bicarbonate or HEPES buffer, which is
serum is normally taken from different types used to maintain a balanced pH in the
of animals such as calf, fetal bovine, horse media.
or human.
To prepare, a “complete media” serum is
Different formulations of basal media are added to the basal media. Antibiotics may be
available for purchase such as Eagle’s added to the complete media to prevent
Minimal Essential Medium (MEM) [Eagle, bacterial growth. Continuous use of
1959], RPMI 1640 [Moore et al., 1967] or antibiotics, however, is not recommended
Dulbecco’s Modification of Eagle’s Medium since it encourages the growth of more
(DMEM) [Dulbecco and Freeman, 1959]. aggressive, resistant bacteria.
Different cell types may prefer different
formulations for optimum growth. Although Alternatively, individual growth factors,
the exact recipes may be slightly different, proteins and other components can be added
all basal media contain the following to the basal media, without the use of serum,
components: in order to prepare the complete media. This
type of a media is said to be “defined
 Carbon source (glucose / glutamine)- media”, since the exact components of the
source of energy factors added are known. Defined-media can

20
be customized and selective for a specific streptomycin (Pen-strep)].
cell type or experimental conditions. The (Optional)
disadvantage of using the defined media is 4. Thaw the fetal-bovine serum, L-
that the preparation can be more time Glutamine and Pen-strep bottles that are
consuming, since each component must be kept in the freezer in the 37oC water bath.
added at proper concentrations, separately. Wipe them down carefully with alcohol and
A number of serum replacements, mix each of them well by swirling the
containing known concentrations of some of bottles a few times, before opening them in
the serum components, are also available the hood.
commercially.
5. Read the formulation of the basal media.
For the cells used in this class we will Eagle’s MEM formulation is appropriate for
prepare undefined media using fetal-bovine growth of most cell types; however, you
serum. To prepare 500ml of complete may have a different, but similar, basal
media: media available to you for your cells. Check
1. Wash your hands before starting. to see if there is any L-Glutamine already
included in the basal media.
2. Turn on the laminar hood and clean it
with 70% ethanol. 6. Uncap the basal media bottle. To the 450
ml, add 50 ml of fetal-bovine serum, using a
3. Gather the materials you need and wipe serological pipette.
them down with alcohol before putting them
in the hood: 7. Using a new pipette, add 5 ml of L-
 Serological pipettes Glutamine to the bottle so the final
concentration of L-Glutamine is 2 mM. If L-
 50ml sterile tubes Glutamine is already included in the basal
media, you do not need to add additional L-
 Bottle of basal media containing Glutamine. Depending on the cell type, the
450 ml. Most manufacturers final concentration of L-Glutamine may be
include 450 ml of basal-media in different (0.5-10 mM).
each bottle to make the
preparations easier. 8. If using antibiotics, add 5 ml of 100X
pen-strep to the bottle. Remember to change
 Bottle of 200 mM L-Glutamine to a new serological pipette for every
(optional) different solution.

 Bottle of fetal-bovine serum 9. Recap all bottles.

 Tube of antibiotics at 100X 10. Mix the bottle by swirling.

concentration [penicillin and

21
11. All reagents used for media preparation
must be sterile. (You can reassure yourself
by filtering the mixed complete media
through a 0.2m filter.)

12. Using a new serological pipette, aliquot

the complete media into 50 ml sterile plastic
tubes.

13. Label each tube of complete-media with

the date and your initials. If antibiotics are
used, note that as well.

14. Cap the tubes and keep them in the

refrigerator. Use one tube at a time.
Complete media can be kept in the
refrigerator for up to a month.

22
II. Routine cell-Culture Subculturing, splitting or passaging is
Maintenance (Feeding and when cells in a culture are divided into two
or more subcultures. Depending on the cells’
Subculturing)
growth rate, cultures may need to be
passaged every 3-7 days.
Once the newly plated cells have attached to
the flask/plate, they will start dividing and
Anchorage-dependent cells in culture are
the cell culture will grow. The cell culture
attached to each other and to the coating on
media should be changed every 2-3 days to
the bottom surface of the flask/plate via cell
provide nutrients to the cells. Changing of
surface glycoproteins. The adhesion
the media is called feeding. When feeding,
molecules are often dependent on Ca2+ for
you need to remove the old media from the
attachment. Subculturing involves removal
culture and replace it with fresh media.
of the media, detachment of cells from the
surface by use of dissociating enzymes and
The rate of growth is usually slow at first
removal of Ca2+ ions by a chelator. Trypsin
after plating, but once the cells have become
is the most commonly used dissociating
accustomed to their new environment, they
enzyme that digests the attachment proteins
will divide rapidly. As cells divide, they will
on the surface of the cells. There are other
cover the bottom surface of the culture
types of dissociating enzymes available that
vessel. The percentage of the surface
may be suitable for a particular cell line,
covered by cells is referred to as
such as collagenase and dispase.
confluency. Anchorage-dependent cells
Dissociating enzymes are often mixed with
keep dividing until the entire 2-dimentional
EDTA (Ethylene diamine tetra acetic acid)
surface available to them is covered (100%
which is a chelator and removes Ca2+ ions.
confluent) and they form a monolayer. The
Long exposure to trypsin is harmful to the
cells will stop dividing as space, nutrients
cells, so cells need to be separated from
and oxygen become insufficient for them, a
trypsin by centrifugation.
phenomenon known as density-dependent
The following is the protocol for routine
growth inhibition. Most cancerous cells
subculturing of a culture growing in a T25
however, may continue to divide on top of
flask using trypsin:
each other and form a multilayer (above
100% confluent). These cultures become
1. Turn on the hood and wipe down with
very unhealthy if they are allowed to
alcohol.
continue dividing when they have
insufficient access to nutrients and oxygen.
2. Gather the material you need and wipe
For all cultures, in order to keep the cells
down with alcohol before placing them in
healthy, it is best to keep them at about 50-
the hood:
80% confluent. This will assure that cells
 Serological pipettes
have enough space, nutrients and oxygen
available to them. Once a culture is above
 Sterile centrifuge tube
75% confluent, it needs to be subcultured.

23
  Tube rack 8. Add 1ml of trypsin to the flask.

 Trypsin/EDTA [trypsin is 9. Lay down the flask and make sure all the
stored in the freezer for long cells are covered by trypsin.
term storage and kept in the
refrigerator for short-term 10. Wait 2-3 minutes. You may want to
storage. Trypsin is an transfer the flask to the incubator to speed
enzyme, and similar to other the process. Trypsin’s enzymatic activity is
proteins, it should not be re- more efficient at 37oC. It is more difficult to
frozen and thawed and should trypsinize some cell lines as compared to
not be kept out of the others. You may have to increase the
refrigerator for too long. Cold amount of trypsin and incubate the flask in
trypsin needs to be warmed the incubator for longer time.
to 37 oC before use.]
11. Observe your cells under the
 Complete media; warmed microscope. Trypsinized cells round up and
are loosened.
 Ca +2/Mg+2 -free Phosphate-
buffered saline (PBS); 12. Tap or rock the flask gently to detach all
warmed. [PBS is an isotonic of the cells.
solution that is often used for
rinsing cells and for routine 13. Once the cells are floating, deactivate
dilutions.] trypsin by adding 4 ml of complete media to
the flask. Trypsin is harmful to the cells, so
 Fresh flasks as soon as cells are detached you should
dilute it by adding media and separate the
3. Observe your cells using an inverted cells from trypsin. Do not leave the cells in
microscope and note the confluency and trypsin for too long.
status of your culture.
14. Transfer the floating cells in trypsin and
4. Inside the hood, aspirate the media from media to a sterile centrifuge tube and label.
the flask.
15. Centrifuge the tube at 100g (about 1000-
5. Transfer about 3 ml of PBS to the flask. 1500 rpm) for 3 minutes.

6. Swirl gently to rinse the cells. 16. Wipe the tube clean and gently transfer
it back to the hood.
7. Aspirate out the PBS. This washing will
remove the residual serum from the flask. 17. Using a sterile glass pipette, aspirate out
Some components of the serum inhibit the the supernatant. Be careful not to remove the
activity of trypsin. cells (pellet).

24
18. Resuspend the cells in 2-5ml of fresh 4 mls of media to the flask to bring up the
complete media. Cells that have been volume to total of 5 mls. So you have
trypsinized and centrifuged tend to clump diluted the cell mixture an additional 5X. the
together. It is important to separate the cells total dilution factor of trypsin, therefore, is
by pipetting up and down a few times. Try (5X) x (5X) = 25X. Twenty five times
to avoid creating too many bubbles. dilution of trypsin is sufficient to deactivate
it and make it harmless to the cells.
19. Transfer the appropriate amount of the
cell suspension into new flasks. (Never re-
use the old flasks).

20. Add enough complete media to the flask

to bring up the volume to 5 ml.

21. Swirl the flask to ensure uniform

distribution of the cells.

22. Observe your cells again to make sure

there are cells floating and there are no big
clumps.

23. Label the flask with your name, date and

the cell-line’s name and place in the
incubator.

24. If there are any left-over cells, aspirate

them into the waste bottle and discard the
old flask.

Notes: The reason behind centrifugation, is

to separate the cells from the harmful
trypsin. Alternatively, you may deactivate
trypsin by diluting it sufficiently by adding
enough media. For most cell lines, if the
volume of trypsin in the media is diluted
enough that it is less than 1/20th of the total
volume, then there is no need to spin the
cells. For example, after initial 5X dilution
of trypsin (step #13 above) by addition of
media and mixing, take out 1ml of the
mixture and transfer it to a fresh flask. Add

25
III. Counting Cells Using a
Hemocytometer 2. Gather the material you need and wipe
them down with alcohol before placing them
in the hood:
Hemocytometer is a device invented by
 Serological pipettes
Louis-Charles Malassez (Verso, 1971). to
count cells. A hemocytometer is made of a
 Hemocytometer
glass slide with a grid on each half (Fig. 10).
Each grid is made of nine squares. Each
square is subdivided into smaller squares.  Micropipettes and tips
The grid is covered by a cover slip and
creates a chamber. Each of the larger  Trypsin; warmed
squares in the grid can hold 0.1l of liquid.
 Ca+2 /Mg+2 – free PBS;
warmed

 Complete media; warmed

3. Remove the media from your flask.

4. Rinse the cells with 3ml of PBS once.

Remove the PBS.

5. Add 1 ml trypsin to the flask and allow 2-

3 minutes for the cells to detach from the
flask. Tap the flask on the side to loosen the
Figure 10. Diagram of a grid of a hemocytometer. cells.
Each square marked by a circle holds 0.1 l of liquid.

6. Observe the cells under the microscope to

In order to count, cells are trypsinized first make sure the cells are all floating.
and a small sample is placed in the
hemocytometer chamber. The cells are 7. Add 4 ml of media to quench trypsin. Mix
visible under the microscope and can be well by pipetting up and down a few times.
counted. Since the volume in each square is
known the concentration of cells can be 8. Using a micropipette transfer 12 l of the
calculated. cell suspension to each chamber of the
hemocytometer. Hold the coverslip on the
To count cells in a T25 using a chamber loosely with your thumb, place the
hemocytometer: pipette tip on the edge of the cover slip and
1. Turn on the hood and wipe down with release the liquid. Repeat for the other half.
alcohol.

26
9. Place the hemocytometer on the stage of a For an explanation of dilution facture see
microscope. Find the grids using the low examples 1-4 on page 31.
magnification lens (4X). Change to higher
magnification (usually 10X is good) to see 13. Clean the hemocytometer and its cover
each square and the cells easily. slip by wiping down with alcohol.

10. Count number of cells in 5 squares of the Notes:

grid (shown in circles on figure 10), that is,  Hemocytometers are fragile and
the four corner squares and the one in the expensive. Please be careful not to drop
middle. Repeat counting five squares for the and break them.
other grid (Count 10 squares total). For
each square, count the cells resting on the  Floating cells settle to the bottom of the
two outside border lines and exclude the flask or tube rapidly and they tend to
cells resting on the inside border lines. For aggregate together and form clumps.
the center square, count the cells resting on Clumps distort the count. It is very
any two of the borders and exclude the cells important to mix the cells well by
on the other two border-lines. Decide on pipetting up and down immediately
which borders you want to count and which before counting to dissociate the clumps
ones are excluded before counting and be and to ensure a homogenous cell
consistent for all your counts. suspension.

11. In order to get an accurate count you  Cells that are outside of the incubator
should count approximately 10 to 50 cells and trypsinized are under stress and may
per square. If you have too many cells, the die if not plated soon. Therefore,
chances of making mistakes are high so you sometimes it may be more important to
need to dilute your sample and repeat speed up the procedure rather than being
counting. If you have too few cells, the accurate. If you are short on time or
number is not statistically valid, and want to speed up the process, you may
therefore, you need to concentrate your cells count cells in five squares rather than
by spinning your cells and resuspending the ten.
pellet in less volume of the media before
counting again.  Observe your cells in culture before
trypsinizing for counting. If you have
12. Calculate cell concentration (cells/ml) very low confluency, you may not get a
using the formula below: large enough count to be accurate. So
you can take out a small sample of cells
(Total # cells counted)(104 )(dilution factor) after they have been trypsinized before
(Total # squares counted) quenching and set aside in a tube for
. counting. Then quench the rest of the
cells in the flask with media. Count the

27
 cells in the tube; since they are more are then trypsinized and pooled together
concentrated, you are more likely to get with the floating dead cells in the same
a large enough number for your count. tube, before counting.
You can always dilute your sample  To distinguish between dead and live
appropriately if the concentration is too cells a stain solution named Trypan-
high (see the examples on page 31). blue may be added to the cells, before
Alternatively, after quenching trypsin if counting. Transfer 50ls of your cells to
you count too little cells, you can a small tube and add 50ls of 0.4%
concentrate the cells and count again. trypan blue. After mixing, pipette 12 ls
Transfer your cells to a centrifuge tube, to each half of a hemocytometer and
spin for 3 minutes, remove the count.
supernatant and resuspend the cells in
less volume of media. Count again. Trypan-blue is a blue stain that can get
inside the dead cells through their porous
 If you have a very confluent flask, you membranes. Live cells don’t allow the
may want to quench trypsin with a larger stain to get in. The dead cells therefore
volume of media to dilute the cells look blue as opposed to the clear live
further. The amount of media depends cells. Once the cells are mixed with
on the confluency of your culture. Note trypan blue they must be counted
that if you add too much media the cells immediately. Live cells will eventually
will become too dilute and obtaining an die in the solution and will turn blue.
accurate count becomes difficult. Keep
in mind, it is always easier to dilute than  To calculate the concentration of the
to concentrate. cells that have been mixed with trypan
blue you must note that the dilution
 Depending on conditions, every cell factor is 2, since the cells were diluted
culture contains a low or high number of 2X with trypan blue solution.
dead cells. Dead cells are usually
detached from the surface and are
floating. When media is removed and
cells are washed with PBS for
trypsinization, most dead cells are
removed from the culture. However, for
some procedures it may be important to
collect and count the number of dead
cells as well as the live ones. To collect
the floating dead cells, the culture’s
media and the PBS used for washing the
cells are transferred to a sterile tube,
before cells are trypsinized. Live cells

28
Example 1: Example 4:

A student counted 80 cells in ten squares of A student has very little cells in her T25.
a hemocytometer. Since he didn’t dilute his She trypsinizes the cells with 1ml trypsin.
culture further the dilution factor is 1. His After the cells are floating she mixes them
culture’s cell concentration is: well and takes out 50 l and sets aside in a
tube. She then adds 4 ml of media to the rest
(80 cells)(104)(1) = 8 x 104 cells/ml of the cells. She puts 12 ls from the tube
(10 squares counted) into each chamber of a hemocytometer and
counts 100 cells in ten squares.
Example 2
(100 cells)(104)(1) = 105 cells/ml
A student has mixed 50ls of her cells with (10 squares counted)
50ls of trypan blue solution. She counts
120 cells in 10 squares of a hemocytometer. The cell suspension in the T25 flask is five
Since she has diluted her cells 2X with times less concentrated:
trypan blue, dilution factor is 2. The cell
concentration of her culture is 105 /5 = 2 x 104 cells/ml

(120 cells)(104)(2) = 2.4 x 105 cells/ml See appendix B for practice problems.
(10 squares counted)

Example 3:

A student counts more than 1000 cells in 10

squares of a hemocytometer. In order to get
a more accurate count, he decides to dilute
his sample before recounting. He takes out
100 ls of his cells and mixes with 900 ls
PBS in a separate tube. After mixing, he
pipettes 12 l in the upper chamber of a
hemocytometer. This time he counts 200
cells in 5 squares. The cell concentration for
his original culture in the T25 flask is

(200 cells)(104)(10) = 4 x 106 cells/ml

(5 squares counted)

29
 Laboratory Exercises 24-well plate with 1 ml of cells at the
concentration of 103cells/ml in each well
and 3 wells at the concentration of 102
Lab exercise 1: Aseptic Technique cells/ml. Use 1ml for each well.
Exercise
Think about your strategy and plan before
starting. You can use the extra wells in the
The objectives of this exercise are to
plate for your dilution steps. Remember, for
practice proper aseptic techniques and
more accurate measurements, not to pipette
become familiar with the concept of serial
volumes less than 50 ls.
dilution.
4. After plating, label the plates and place
In this exercise you will pipette the less
them in the 37oC incubator.
expensive bacterial media into cell culture
plates, practicing the use of aseptic
5. Clean the hood and wipe with alcohol
techniques and the use of serological and
before turning it off.
micropipettes. You will dilute water in LB
(Luria Bertani) media 103and 104 times
6. After a week, observe your plate. If there
(refer to Appendix A for a review of serial
are bacteria or fungus growing, it means that
dilutions). Note that water (or PBS) is being
you did not use good aseptic techniques. If
diluted in LB. You will pretend that water
the media looks clear, then good aseptic
(or PBS) is the cell suspension of 106
techniques were used.
cells/ml and bacteria growth medium (LB) is
the cell culture medium.

1. Turn on the laminar flow hood and wipe it

with alcohol.

2. Gather the material you need and wipe

them down with alcohol before placing them
in the hood:

 Serological pipettes

 10 mls of LB

 1ml of sterile dH2O

 Tube rack

 24-well cell culture plate

3. Pretend that the water you are given is a

cell suspension at the concentration of 106
cells/ml. You are asked to plate 3 wells of a

30
Lab exercise 2: Plating Cells from note of the freezing date and other
Frozen Stocks information on the vial.

When cells are frozen they are kept in 4. Place the frozen vial in a small container
compete media plus 5-15% DMSO in the 37oC water bath. Be careful that the
(dimethyl sulfoxide). DMSO prevents cap does not get wet to avoid possible
crystallization and breakage of the frozen contamination.
cellular components. Although beneficial to
preserve the frozen cells, DMSO is harmful 5. As soon as the vial is defrosted (about 1-2
to cells while growing in culture. Therefore, minutes) take it out of the water bath and
after defrosting, cells are centrifuged to wipe down well with alcohol. Don’t let the
separate them from the DMSO before they defrosted cells sit in DMSO for too long.
are plated. When cells are defrosted they
must be plated immediately. You should 6. Mix the contents of the vial by inverting
organize your work area and be ready to the tube several times, and take it to the
proceed with the plating before you take out hood.
a frozen stock.
7. Transfer 9mls of the complete media to
1. Turn on the laminar-flow hood and wipe the 15ml tube.
down with alcohol.
8. Using a sterile serological pipette or a
2. Gather your material and wipe down with micropipette, transfer the contents of the vial
alcohol before putting them in the hood: (usually 1ml) to the same 15ml tube. This
will dilute the amount of DMSO ten times
 One 15-ml sterile centrifuge (10X dilution).
tube
9. To wash and remove any remaining cells
 Tube rack in the vial, pipette 1ml of the contents from
the tube back into the vial and then pipette it
 Serological pipettes back in the tube.

10. Close the top of the tube and label.

 Complete media warmed up
at 37oC
11. Using a balanced tube (see page 10),
centrifuge the cells at about 100g (about
 One T-25 cell culture flask
1000-1500 rpm for most clinical
centrifuges) for about 3 minutes.
3. Take out a frozen vial of cells from the
-80 0C freezer or the liquid nitrogen tank.
12. The heavy cells will form a pellet and
Always look at the label of the vial to ensure
are separated from the media (supernatant).
you have the correct cell line and make a

31
The pellet may not be visible to the eyes. Cells my take up to about a day to attach to
The pellet may be found on the side of the the bottom of the flask and start dividing.
tube close to the bottom. Most of the cells may not survive the
process of freezing and defrosting and may
13. Take out the tube gently. Shaking the never attach to the flask. However, if the
tube will resuspend the cells. Wipe down the number of cells is high enough, even a small
tube with alcohol and put it back in the percentage of cells that survive are sufficient
hood. to start a new culture.

14. Using the aspirator in the hood and a

sterile glass pipette, remove the supernatant
from the tube carefully. To avoid sucking
up the pellet, you may leave a small amount
(~500µl) of the supernatant behind.

15. Transfer 3mls of complete media to a T-

25.

16. Transfer 2mls of complete media to the

pellet and resuspend the pellet by pipetting
up and down a few times. Although it is not
possible to avoid bubbles, pipette gently to
reduce the formation of the bubbles.

17. Transfer the resuspended cells to the

T25. Mix by pipetting up and down a few
times. Close the flask’s cap.

18. Swirl the flask gently. Be careful not to

get the flask’s cap wet.

19. Observe the cells using an inverted

microscope. You should have plenty of cells
floating in the media.

20. Place the flask in the incubator.

(Remember to loosen the cap if the flask is
not vented).

32
Lab exercise 3: Count and Plate 104 6. Add 1ml trypsin to the flask. Make sure
Cells in a T25 all of the cells are covered by trypsin.

The objectives of this exercise are to 7. Wait about 2-3 minutes for the cells to
practice trypsinizing, counting and plating a detach.
specific number of cells. Please read all of
the protocol before starting. 8. Tap the side of the flask and observe
under the microscope to make sure all of the
1. Turn on the hood and wipe down with cells are floating.
alcohol.
9. After cells are detached, quench trypsin
2. Gather the material you need and wipe by adding 4 ml of media.
down with alcohol before placing them in
the hood: 10. Mix well by pipetting up and down a
 Serological pipettes few times.

 Micropipette tips Count with a hemocytometer

 Microfuge tubes 11. Take out 50 l of the cells to a

microfuge tube.
 Ca+2 /Mg+2 –free PBS,
warmed 12. Add 50 l of trypan blue solution to the
tube and mix.
 Trypsin, warmed
13. Transfer 12ls of the cell suspension in
 Complete media, warmed the tube to one half of the hemocytometer.
Repeat for the other half.
 Trypan blue
14. Count the number of live cells in 10
3. Observe your cells and make sure they are squares. If the number is too high (>500),
healthy. Note the confluency of your flask. you need to dilute your cells appropriately
and count again. If the number is too low
Trypsinize (<50), you need to concentrate your cells by
transferring to a centrifuge tube, spinning,
4. Inside the hood remove the media from removing the supernatant and resuspending
your flask. the cells in less volume of complete media.

5. Wash the cells with 3 ml of PBS. Remove 15. Calculate cell concentration and total
PBS. This washes the serum away. number of cells.

33
 # of cells in 10 squares 19. Calculate the amount of fresh media for
Dilution factor for mixing the final cell suspension and pipette it inside
with Trypan blue a fresh T25 flask.
Further dilution factors, if
any
Total dilution factor V1 (calculated above)
Concentration of cells in Volume of fresh media
the flask (cells/ml)- C1 Total volume 5 mls
Total volume in the flask
Total number of cells 20. Take out the correct volume (V1) of your
culture and add it to the media inside the
Plate new flask.

16. You are planning to plate 10, 000 cells 21. Mix by pipetting.
in a T25. Normally 5 ml of media is
sufficient for a T25 flask. So you need to 22. Label the flask, observe it under the
make a suspension of 10,000 cells in 5ml or microscope and place it in the incubator.
2000 cells/ml (final concentration).
Calculate the volume that you need to take 23. You may plate more than one plate for
out of your culture which contains 10,000 future use or discard the remaining cells by
cells using the formula (C1)(V1)= (C2)(V2), aspirating inside the waste bottle and
where C1 is your culture’s concentration that disposing of the flask appropriately.
you have calculated. V1 is the unknown
volume containing 10,000 cells. C2 is the 24. Observe the cells after they are settled
final concentration of 2000 cells/ml and V2 next time you are in class and note the status
is the final volume that is 5 ml. Solve for V1. of the cells.

C1 (initial concentration, Analysis questions:

calculated above)
C2 (final desired concentration)
V2 (final desired volume) 1. What was the status of your cells before
V1 (initial volume of culture to starting? (% Confluency, color of the media
start with, unknown) and general health of the culture.)

17. If V1 is less than 50 l, then go back to 2. Did you have enough cells to plate 104
your original culture and dilute some of it cells? If so how many cells remained after
appropriately in order to get a larger V1. plating?
(Refer to appendix A for serial dilutions).
3. Did you have to dilute your culture
18. Mix your culture well since cells might further or concentrate it for more accurate
have settled and might have formed clumps. count? If so how did you do it?

34
4. For step 17, did you have to dilute your
culture to increase the V1 value? How did
you do it?

5. What is the status of your cells after

settling (step 24)?

35
Lab Exercise 4: Survival Assay- number of colonies represents cell survival
Sensitivity to UV Exposure and the size of colonies represents growth
rate. The bigger colony size can be
interpreted as faster growth rate.
After plating, cells attach to the surface of
the culture vessel and start dividing. A
Plating efficiency is one of the techniques
single cell divides into two daughter cells
often used for cytotoxic studies. Cytotoxic
which divide into four and then eight and so
studies involve measurement of altered
forth, thus forming a colony. A colony
metabolism or loss of viability due to a toxic
refers to a population of cells that are all
factor. In vitro cytotoxicity studies are often
descendents of a single parental cell. Cells in
used by environmental scientists and
a colony sit close to each other and if there
pharmaceutical companies to screen for
are enough cells in a colony, they can easily
potentially toxic drugs and environmental
be seen without the help of the microscope.
factors. Plating efficiency is a form of a
cytotoxicity assay that measures cell
When plating, many of the cells die in the
viability several divisions after exposure to
process and never reattach to the flask.
the toxic reagent. To study the effects of a
Among those who do attach, some may die
toxic reagent or drugs on cell survival, cells
later. Some of the cells may stay alive but
are plated at low densities in separate
will not divide enough times to make a
vessels and exposed to different dosages of
colony. Plating efficiency (or cloning
the reagent. After several divisions, the
efficiency) is a measurement used to
number of colonies is counted. PE and
identify the percentage of cells that survive
surviving fraction (SF) values are
and are able to divide and form colonies
compared among cells that were exposed to
after being plated. Plating efficiency is a
different dosages and control cells with no
value that measures cell survival when cells
exposure.
are plated at low densities (2-50 cells/cm2).
When cells are plated at lower densities,
they are far apart and are not able to assist
each other for survival. These cells are on
their own, and if they survive they will The lower SF values represent less survival
divide and form colonies. Each colony as a result of exposure. In addition, average
represents a single cell that has managed to colony size can reveal the effect of the toxic
survive. To measure plating efficiency, cells reagent on growth rate. The SF values can
are plated at low densities and allowed to be plotted against dosages to generate a
grow 7-10 days. During this time the cells survival curve.
that have survived will divide and form
colonies. The colonies are then counted and In the following exercise you will determine
plating efficiency (PE) is calculated: the PE and SF values for cells that have
been exposed to different dosages of UV
(ultra violet) light. The objective is to
determine the level of sensitivity of your
Plating efficiency is used to study cells to UV light. You may do the
differences in cell survival and growth rate experiment with two or more cell lines and
within cells of the same population or compare their UV sensitivity levels.
among cells of different populations. The

36
Ultraviolet light exposure causes formation  Serological pipettes
of covalent bonds between two adjacent
pyrimidines (C and T) in cellular DNA  Micropipette tips
forming pyrimidine dimers (Goodsell,
2001). Pyrimidine dimers block progression  Sterile centrifuge tubes
of replication forks and, if left unrepaired,
may lead to base deletions or substitutions  Ca+2 /Mg+2 PBS-warmed
and cause mutations. The mutations are
subsequently propagated as the cell goes
 Trypsin- warmed
through more divisions and can have
disastrous effects for the organism (for
 Hemocytometer
example, skin cancer). Fortunately, our cells
are equipped with a number of proteins that
are specialized in recognition and repair of  Five 6-well plates
pyrimidine dimers. Mutations in the genes
that code for these proteins can make the 3. Observe the cells under the microscope
repair mechanism inefficient and cause and note the confluency of your cultures.
devastating diseases. People who are Work with one cell line at a time.
affected by these mutations are very
sensitive to light exposure and develop 4. Remove the media, wash with PBS and
severe sunburns and tumors and die at trypsinize cells with 1 ml trypsin. (Refer to
young ages. the page 34 for detailed protocol of
trypsinizing.)
In the following exercise you will compare
the sensitivity levels of two cell lines to UV 5. After cells are detached and floating, add
radiation. The following exercise will be 4mls of media and mix by pipetting.
done over a week. On the first day, you will
plate your cells at low density in multi-well 6. Using a hemocytometer count the number
plates. After one or two days, the cells are of cells and calculate cell concentration.
exposed to several dosages of UV light. The (Refer to page 26, counting cells using a
cells are allowed to divide and form colonies hemocytometer, for a detailed protocol).
for one week. The colonies are then stained
and counted. Finally, you will draw a 7. Plate 200 cells into one row or 3 wells of
survival curve and determine the sensitivity the six-well plate (density of about 20
of your cells to UV light. Some of the steps cells/cm2) according to the diagram in figure
may be performed by the instructor or the 12. Repeat for the other four plates. (The
lab aids. number of cells that you need to plate may
be different depending on the plating
efficiency of your cells). When plating cells
Plating of the cells
in multi-well plates, it is best to prepare a
1. Turn on the hood and wipe down with master-mix first and then aliquot into the
alcohol. wells rather than plating each well
separately. This is to reduce the chances of
2. Gather the material you need and wipe mistakes in plating each well and keeping
them down with alcohol before placing them the wells consistent. For this experiment we
in the hood: will plate three wells per plate for each cell
line, which is a total of 15 wells. You can

37
use 2 ml of media for each well. You must
always prepare a master-mix with slightly
larger volume than you need to account for
pipetting errors. Therefore, make 32 ml of
the master-mix with 2 ml extra. Calculate
the volume of cells you need to take out in
order to make your master-mix (V1). Use the
(C1)(V1)= (C2)(V2) formula.

Figure 11- Diagram of the six-well plates. Seed 3

Cell Cell wells (or a row) of each plate with each cell line.
line 1 line 2
# of cells counted in 10
squares of the
hemocytometer
Dilution factor for counting
Cell concentration, C1 UV exposure
Final cell concentration of
the master-mix (cells/ml), C2 One day after plating:
Final volume of the master 32 ml 32 ml
mix (ml), V2 13. Turn on the hood and wipe down with
Initial volume of the culture, alcohol. Gather you material and wipe down
V1 with alcohol:
Volume of complete media
 Serological pipettes

8. Prepare the master-mix by mixing the  PBS (with Ca+2 /Mg+2)

appropriate volume (V1) of cells from your
culture to a large tube with enough complete  Complete media, warmed
media to bring up the volume to 32 ml.
 UV protection goggles
9. Mix well by pipetting.

10. Aliquot 2 ml into each well. 14. Remove the media from your wells.

11. Label the plates as shown in (Fig. 11) 15. Wash the wells with 0.5 ml of PBS per
and put them in the incubator. well. Remove the PBS such that the wells
are dry.
12. If you are using more than one cell line,
repeat the above procedure for the other cell
line, using the second set of three wells on 16. Expose each of the plates to one of the
the six-well plates. following doses: 0, 2, 4, 8 or 10 seconds of
UV light. To expose the plates:

 Wear protective goggles.

38
  Remove all other cells that 22. Allow the cells to stain at room
you are NOT treating with temperature for about 30-60 min.
UV from under the hood. 23. Transfer the stain to the waste bottle (the
stain can be filtered and re-used). Do not use
 Place the plate to be treated the aspirator: crystal violet will stain the
under the UV light. tubing of the aspirator, which is difficult to
clean.
 Remove the lid from the
cells that you are going to 24. Gently pour some tap water into each
treat with UV. well and wash the excess stain 2-3 times.
Dump the water waste in the sink with
 Turn on the UV lamp for 0, running water.
2, 4, 8 and 10 seconds.
25. Invert the plates and let them dry.
17. After UV treatment, quickly add 2 ml of
fresh complete media per well. 26. Count number of colonies for each well.
Colonies that are too tiny (less than 50 cells)
18. Put the plates back in the incubator and are not counted. Different sizes of colonies
do not move for 7-10 days. represent variations in growth rates among
cells in the same culture.
Counting colonies
27. Calculate the average number of
In order to see the colonies clearly, you will colonies for each set of three wells with the
stain the colonies with a blue/violet stain same treatment.
called crystal violet. Crystal violet stain is
mixed with methanol, which is a fixative. 28. Calculate PE and surviving fraction
Methanol will kill the cells but keeps the cell values for each dosage and each cell line.
structures intact and prevents disintegration
of the cells. 29. Graph a surviving curve and analyze
your data.
19. Aspirate out the media.
Analysis questions:
20. Wash the cells once with about 0.5-1 ml
per well of PBS (with Ca+2 /Mg+2). Remove 1. Count the average number of colonies for
the PBS. each set of three wells and calculate the PE
value for each dosage? Repeat for each cell
21. Add 0.5 ml of Crystal violet/Methanol to line. How do the PE values change with
each well. different dosages?

39
 IC50 means higher sensitivity. Which cell-
Cell-line 1 Cell-line 2 line is more sensitive to UV radiation?
Dosage Ave. # of PE Ave. # of PE
(sec) colonies colonies 6. If you were able to repeat the experiment,
0
how would you do it differently?
2
4
8 7. Discuss your results further in your
10 notebook.

2. Calculate the SF for each dosage and each

cell line. How do the SF values change with
different dosages?

Dosage SF Cell-line 1 SF Cell-line 2

(sec)
0 1 1
2
4
8
10

3. How do the average sizes of colonies

differ with different dosages of UV? What
do the differences in colony size indicate
about the effect of UV on your cells?

4. Draw a survival curve for this experiment.

Survival curves are drawn in a semi-log
graph format, with the SF values on the Y-
axis in the log format and dosage on the X-
axis in standard format. Draw both curves
for the two different cell lines on the same
graph.

5. What is the IC50 value for each cell line?

IC50 is the dosage at which there is 50%
inhibition of colony formation (SF value of
0.5). IC50 values are often used to compare
sensitivities to toxic reagents. Higher IC50
values indicate lower sensitivity and lower

40
Lab Exercise 5: Live and Dead Cell cells do not have functional esterases and so
Identification by Using Propidium will not be green.
Iodide and Calcein AM
Day 1- plating: (This can be done by your
instructor or the lab technicians)
In this exercise you will use fluorescent
molecules to distinguish between live and
1. Plate cells in 24-well plates (Fig. 12) so
dead cells. Fluorescent molecules, also
the wells are 60-80% confluent for the day
known as fluorophores, can absorb the
of the experiment. Use 1ml of media per
energy of light at specific wavelengths and
well.
emit less energetic fluorescent light. When a
fluorophore absorbs the energy of light it
becomes excited by elevating to a higher
energy level (excitation). The excited
fluorophores go back to the ground state by
releasing the energy in form of heat and
emitted light (emission). A large variety of
fluorophores have been made and modified
to interact with specific cellular structures in
order to study them.

Propidium Iodide (PI) is a fluorescent

molecule that absorbs light most efficiently
at 500-550 nm and emits bright red
Figure 12- Diagram of the 24-well plate. One half of
fluorescent light at 600-650 nm
the plate marked by X is not used for the experiment.
wavelengths. Live cells are impermeable to
PI, so PI can only enter dead cells that have Day 2:
porous membranes. Once inside the cell, it
intercalates between nucleic acids (both 2. Your instructor will expose the first two
RNA and DNA). PI is considered hazardous rows of the cells to UV radiation (or other
and you must wear gloves when working toxic reagents) prior to the start of the class.
with PI.
The following steps are performed by the
Calcein AM is a derivate of calcein and is students:
not fluorescent. Calcein AM is permeable to
both live and dead cells. Once inside the live 3. Turn on the hood and wipe it down with
cells, esterase enzymes convert calcein AM alcohol.
into calcein. Calcein is a bright green
fluorescence compound that absorbs and 4. Gather your material and wipe down with
emits light at 495/520 nm respectively. Dead alcohol before placing them in the hood:

41
  Micropipettes and tips 12. Remove the media from the wells in the
first two columns of the plate labeled CAM
 Serological pipettes and CAM/PI.

 70% methanol in dH2O 13. Add 1 ml of media to the third well of

the third row.
 PBS (with Ca+2/Mg+2), warmed
14. Rinse all of the cells in the first two
 Complete media, warmed columns with 0.5 mls/well of PBS twice.
Remove PBS.
 10 g/ml Propidium Iodide (PI)
15. Wash one more time with PBS and leave
the PBS in for 5 min.
 Calcein AM

16. Remove PBS from all of the wells in the

5. Observe the cells under the microscope.
first two columns.
Do you see a difference between the rows of
cells that were exposed to UV (or other
reagents) and cells in the other rows? 17. Add 200 l per well of 8M Calcein
AM per well of the first and second column
6. Remove the media from the third row of of cells.
cells.
18. Put the plate back in the incubator for 30
7. Rinse the cells in the third row with 0.5 minutes.
ml of PBS and remove PBS. Repeat the
wash. 19. Remove the Calcein AM and media
from all of the wells in all the rows and
8. Wash one more time with PBS, this time columns.
leave the PBS in for 5 min.
20. Rinse all of the wells with PBS once.
9. Remove PBS. Keep the PBS in the first column but remove
it from the second and third columns.
10. Add 0.5 ml per well of 70% methanol to
the third row and incubate for 30 min. 21. Add 200 µl per well of g/ml PI to
Methanol will kill and fix the cells so the the wells of the second and third columns.
cellular structures remain intact after dying. Incubate at room temperature for 1 min.

11. Remove the methanol from the third 22. Examine the cells under the fluorescence
row. microscope. Use the green filter cube to see
the red color of PI and the blue filter cube to
see the green color of Calcein.

42
23. Compare the fluorescent colors in
different wells and write your observations
in your notebook.

Analysis questions:

1. What color fluorescence do you see for

each row and each column? Is what you see
the same as you expected? Why?

2. Is there a difference between the row of

cells exposed to high level of UV and the
row of cells exposed to the low level of UV?

3. What is the pattern of red fluorescence

that you observe for the cells stained with
PI? Explain which cellular structures are
red?

4. Do you recognize any pattern of green

fluorescence for the cells that are stained
with calcein?

5. Discuss the results in your notebook.

43
Lab Exercise 6: Transfection are assayed 48-72 hours post transfection,
before the loss of DNA. Occasionally, the
Transfection is a method used to transfer introduced DNA is inserted into the host
non-viral recombinant DNA into genome. The DNA becomes part of the host
mammalian cells. Transfer of viral genetic genome and is passed to the next
material into eukaryotic cells is referred to generations. This type of transfection is
as infection. Transfected cells are used to known as stable transfection. The cells that
study gene expression and regulation, are stably transfected need to be selected for
protein expression and function, gene and separated from the transient or non-
therapy and more. DNA sequences of transfected cells.
interest are inserted into carrier DNA
molecules; commonly known as vectors, by In the following exercise, you will transfect
the use of recombinant DNA techniques. your cells transiently by using two different
One of the most commonly used vectors for plasmids:
transfection is a plasmid. Plasmids are small
circular DNA molecules that naturally exist 1. pDsRed2-Mito vector (from Clontech
in bacteria and some eukaryotic cells. Laboratories Inc.
Plasmids are engineered and designed to http://www.clontech.com/), carrying the
carry sequences that enable the plasmids to sequence coding for a fusion protein of a
be replicated by the host cell’s replication red fluorescent protein and a mitochondrial
machinery. There are various methods used targeting sequence.
to introduce the vector, which is carrying the
DNA sequence of interest into the cells. 2. pEGFP-Actin vector (from Clontech
Examples of these methods for introducing Laboratories Inc.), carrying the sequence for
DNA into cells include the use of electric a fusion protein, consisting of green
currents, microinjection, shooting of DNA fluorescence protein (GFP) and cytoplasmic
coated particles into cells, and forming a -actin.
complex of DNA and cationic molecules
that can enter the cell by endocytosis or In the following protocol Fugene® HD
membrane fusion. If there are sequences transfection reagent (from Promega
that are recognized by the host cell present, http://www.promega.com/tbs/tm328/tm328.
after entering the nucleus, the genes that are html) is used to insert the plasmid DNA into
carried on the foreign DNA may be the cells. You may have a different
transcribed and expressed into proteins by transfection reagent available and need to
the cell. For most transfections, the foreign follow the manufacturer’s protocol. Note
DNA molecule stays independent of the host that you may need to optimize the protocol
genome and is eventually lost during for your cell line and DNA preparations.
subsequent cell divisions. This type of
transfection in said to be transient. In order Two days after transfection, the cells are
to study transient transfections, the cells fixed with 4% paraformaldehyde in order to

44
keep the cellular structures intact. The cells 6. Dilute your DNA in serum-free media.
are then observed by using a fluorescent Prepare 4 tubes:
microscope.
1 2 3 4
Control Mito Actin Mito/
Day 1- plating: (This can be done by your
Actin
instructor or the lab technicians). Serum- 100 l 98 l 98 l 98 l
free
media
1. Plate cells in 24-well plates, so the wells pDsRed2- 0 2 l 0 1 l
are 70-80% confluent by the day of the Mito
experiment. Use 1ml of media per well. (1g/l)
pEGFP- 0 0 2 l
Actin l
Day 2-Transfection: (1g/l)

2. Turn on the hood and wipe down with 7. Add 8 ls of Fugene® HD to each of the
alcohol. mixtures (8:2 ratio of Fugene: DNA). Mix
by vortexing and incubate at room
3. Gather the material you need and wipe
temperature for 15 minutes.
them down with alcohol before placing them
in the hood:
8. While waiting, change the media in your
 Pipettors plate. Add 1mls of fresh media per well.

 Pipette tips 9. Add 25 ls of the transfection mixture to

each well drop-by-drop, according to the
 Basal media (serum-free)
diagram in figure 13. Note that there are
three wells for each mixture. Discard the
 Complete media, warmed
extra mixture.
 Fugene® HD
10. Mix the plate by moving it back and
 Vector DNA (1g/l), mix forth or left and right gently, without spilling
before use the media.

 Sterile microfuge tubes 11. Place the plate in the incubator for 2
days.
4. Observe the cells before the start of the
procedure. The cells should be healthy and
actively dividing.

5. Mix the vial containing Fugene® HD by

vortexing or manually inverting and bring it
to room temperature.

45
 18. Wash the cells with 0.5 mls of PBS per
well. Remove PBS.

19. Add another 0.5 ml of PBS per well and,

this time, keep the PBS in to prevent the
cells from drying out.

20. At this point you can store the plates in

the refrigerator for future viewing.
Figure 13- Diagram of the 24-well plate. Half of the
plate marked by X is not used for the experiment. Fluorescent molecules are photosensitive, so
the plate should be kept in the dark.
Day 4- Fixing:
12. 48 hours after transfection, turn on the View the cells:
hood, wipe down with alcohol and gather
your material (wipe down with alcohol): 21. Place the plates on a fluorescent
 Serological pipettes microscope and observe the results. You
need the green filter-cube to see the red
 PBS (with Ca+2/Mg+2), fluorescence and the blue filter cube to see
warmed the green fluorescence.

 4% paraformaldehyde in 22. Note your observations in your

PBS, wear gloves notebook.

 Paraformaldehyde waste Analysis questions:

bottle
1. Do you see a difference in fluorescence
13. Remove the media from your cells. between the control cells and the transfected
cells? Are your observations as expected?
14. Add 0.5 ml of PBS per well and remove Explain.
PBS.

15. Add 0.5 ml of 4% paraformaldehyde per 2. What percentage of cells do you estimate
well. are transfected with pDsRed2-Mito?
Approximately what percentage of the cells
16. Wait 15 minutes at room temperature. are transfected with pEGFP-Actin? Which
plasmid has a better transfection efficiency?
17. Transfer the paraformaldehyde to the
waste bottle (Paraformaldehyde is toxic and
should not be discarded in the sink). 3. Do you recognize any pattern in
fluorescence of the cells transfected with

46
pDsRed2-Mito (where do you see the
fluorescence)? How about pEGFP-Actin?

4. Discuss your results in your note book.

47
Lab Exercise 7: Freezing Cells To freeze cells:

1. Turn on the hood and wipe down with

When freezing, cells are resuspended in
alcohol.
freezing media which is complete media
plus 10% DMSO (Dimethyl sulfoxide). 2. Gather the material you need and wipe
DMSO prevents crystallization and damage them down with alcohol before placing them
to the cells. To keep the cells viable, they in the hood:
need to be frozen slowly. The cells are  Serological pipettes
transferred to cryo-vials and placed in
freezing boxes. Freezing boxes are filled  Centrifuge tubes
with isopropanol to keep the cells from
 Tube rack
freezing too fast (Fig. 14). When the box is
placed in the -80oC freezer, the temperature  Trypsin, warmed
goes down at the rate of 1oC per minute. For
long-term storage, frozen vials are  PBS (Ca+2/Mg+2- free),
transferred from the -80oC freezer to a liquid warmed
nitrogen tank.
 Complete media, warmed

 DMSO, wear gloves

 Cryovials

 Hemocytometer

3. Observe your cells first. Your culture

must be healthy and more than 60%
confluent to be suitable for freezing.

4. In a sterile tube, prepare freezing media

Figure 14. Cell culture freezing box and cryovials. by mixing 9mls of complete media with 1
ml of DMSO.
Many of the cells die in the process of
freezing and defrosting, therefore, usually 5. Remove the media from your culture.
cells are frozen at very high concentrations
so, when defrosted, there is a good chance 6. Wash the cells with 3mls of PBS and
that some cells will survive and make a remove the PBS.
culture. A concentration of 1 x 106 cells/ml
is usually used for freezing. Wear gloves 7. Add 1ml trypsin to the flask. Make sure
when working with DMSO. the cells are covered by trypsin.

8. Wait 2-3 minutes for the cells to detach.

48
9. Tap the side of the flask, and observe 23. Put the box in the -80oC freezer. (Cells
under the microscope to make sure all of the may be kept in the -80oC freezer for up to a
cells are floating. few weeks, depending on the cell type.)

10. Quench trypsin by adding 4mls of 24. After one day you can transfer the tubes
complete media and mix well by pipetting to a liquid Nitrogen tank to keep
up and down. indefinitely.

11. Pipette 12ls on each side of a # of cells in 10 squares of the

hemocytometer and count cells. hemocytometer
Cell concentration
Total number of cells
12. Calculate cell concentration in your
Volume of the freezing media
culture.

13. Calculate total number of cells in your

culture: Notes:
(Total cell #)= (cells/ml)(total volume, mls)
 You can use Styrofoam boxes instead of
14. Transfer the cells to a sterile centrifuge the isopropanol boxes for freezing the
tube. cells. Place the tubes in Styrofoam
boxes. Make sure they are covered on all
15. Spin the cells at about 1000-1500 rpms sides. Label with your name, date and
for 3 min. cell type. After you are finished put it in
the -80oC freezer.
16. Wipe the tube down with alcohol and
place it back in the hood.  For routine freezing you can estimate the
number of cells without counting. For a
17. Aspirate out the supernatant. Be careful 100% confluent T25 you can generally
not to aspirate the cells in the pellet. use 4mls of freezing media and freeze
four vials. (Note that for different cell
18. Add the appropriate volume of freezing lines this number may be different).
media to the pellet to get concentration of
1 x 106 cells/ml.  Freezing media is kept in the
refrigerator.
19. Resuspend the cells in the freezing
media by pipetting up and down a few  DMSO is light sensitive. Cover the tube
times. of freezing media with aluminum foil to
keep it in the dark.
20. Transfer to cryotubes (up to 1ml per
tube).

21. Label the tubes with your name, cell

type and date.

22. Place the tubes in isopropanol freezing

boxes.

49
Lab Exercise 8: Stem Cell types. Half of the cells will be differentiated
Differentiation spontaneously into cardiomyocytes (heart
muscle cells) and the other half will be
directed to differentiate into neurons.
Stem cells are cells that are able to renew
themselves (self-renewal) indefinitely,
The cells are grown in uncoated Petri dishes
while producing cell progeny that can
to prevent adhesion of the cells to the plate.
mature into specialized cells
The cells growing in suspension form
(differentiation). Adult stem cells in our
embryoid bodies (EB). The EBs are then
body are able to regenerate cells and renew
plated on gelatin-coated plates to adhere to
damaged tissues. Embryonic stem (ES)
the plates and continue differentiation. In
cells are a type of stem cells that are formed
order to direct cells into neuron
very early during embryonic development.
development, retinoic acid is added to the
Embryonic stem cells are said to be
media. Retinoic acid is a signaling molecule
pluripotent, meaning that under proper
that has an important role in neural
conditions they can generate all three cell
development and activity at the embryonic
layers (ectoderm, mesoderm and endoderm).
stages (Dhara and Stice, 2008).
ES cells are maintained as a monolayer
Since you are not here every day, some of
adhered to the culture vessel. The recipe for
the steps will have to be done by the lab
stem cell culture media contains important
technicians.
components to keep the cells from
differentiation and to preserve the
Day 1- Plating cells in suspension
pluripotent characteristic of the stem cells
(pluripotent media). To trigger 1. Turn on the hood and wipe it down with
differentiation, ES cells are grown in the alcohol.
differentiation media that allows cells to
differentiate. The cells are plated on 2. Gather your material and wipe down with
uncoated plates to prevent adhesion to the alcohol before placing them in the hood:
plates. The cells in suspension form
 Serological pipettes
aggregates (spheres) called embryoid
bodies (EB); and differentiation initiates  PBS (Ca+2/Mg+2-free),
spontaneously upon aggregation of cells. warmed
The cells can potentially differentiate into all
cell types spontaneously. Several methods  Trypsin, warmed
have been developed to direct differentiation
into specific cell types by culturing stem
cells under appropriate and controlled  Embryonic stem cell
differentiation media,
conditions. In the following exercise warmed
(adopted from a protocol by Dr. J. Ng) you
will differentiate ES cells into specific cell  A hemocytometer

50
  Centrifuge tube and tube rack 17. Resuspend the cells in the appropriate
volume of differentiation media (10 mls for
 Sterile 10-cm bacteria culture each plate to the concentration of 300,000
dish cells/ml).

3. You will be given a T25 flask of 18. Seed 10 ml of the cells onto each 10-cm
pluripotent ES cells. Petri dish.

4. Observe the cells using a microscope. 19. Incubate the dishes in the 37°C
incubator.
5. Aspirate medium off.
Day 2 or 3- Feeding the embryoid bodies
6. Wash with 5 ml of PBS.
20. Turn on your hood and wipe it down
7. Rock the flask gently and aspirate PBS. with alcohol.

8. Repeat steps 6 and 7. 21. Gather your material and wipe them
down with alcohol before placing them in
9. Add 1 ml of 0.5% trypsin to cover the the hood:
cells.
 Serological pipettes
o
10. Incubate the flask in 37 C incubator for
1-4 minutes or until cells are uniformly  15-ml conical tubes
dispersed into small clumps.
 Tube rack
11. Tap sides of flask gently to dislodge the
cells.  Differentiation media,
warmed
12. Add 5 ml of ESC differentiation media
to inactivate the trypsin.  Sterile 10-cm bacteria Petri
dish
13. Count the cells with a hemocytometer.
22. Observe your cells under the
14. You need to prepare 10mls of 3 x 10 5 microscope. By now the cells must have
cells/ml suspension in ESC differentiation aggregated together and formed floating
media to be plated in one 10-cm bacteria embryoid bodies (EB).
Petri dish. This means that you need 3 x 10 6
cells total for each dish. Therefore, transfer 23. Carefully transfer all the media plus
the appropriate number of cells for two embryoid bodies from each dish into a
plates (6 x 106 cells) to two centrifuge tubes. separate sterile 15ml tube.

15. Spin the cells at 100 g (about 1000 rpm) 24. Wash each dish with 5mls of
on a clinical centrifuge. differentiation media and add it to the tube.

16. Remove the supernatant. 25. Close the caps and transfer the tubes to

51
the incubator inside a clean rack.
26. Allow embryoid bodies to settle to the 35. Allow the embryoid bodies to settle to
bottom of the tubes for 10 minutes. the bottom of the tubes for 10 minutes.

27. Transfer the tubes gently back to the 36. Carefully aspirate off the media, leaving
hood and carefully aspirate off the media, the EBs undisturbed.
leaving the EBs undisturbed.
37. Prepare 10 ml of differentiation media
28. Add 10mls of fresh ESC differentiation plus 5 M retinoic acid by adding 10 l of
media to each tube. (Do not pipette up and themM stock solution.
down, it may break the EBs)
38. Add the differentiation media plus
29. Using a serological pipette, gently retinoic acid mixture to one of the tubes to
transfer the EBs back to new 10-cm Petri direct the cells into neuron differentiation.
dish.
39. Transfer the EBs plus the differentiation
30. Place the dishes back in the 37°C media and retinoic acid to a new10-cm Petri
incubator. dish.

Day 4 or 5- Feeding and directing 40. Add10 ml of plain differentiation media

differentiation to the second sterile tube (for
cardiomyocytes differentiation). And
31. Turn on and wipe your hood down with transfer to a separate 10-cm Petri dish.
alcohol.
41. Label the dishes and incubate them back
32. Gather and wipe down your material in the 37°C incubator.
with alcohol:
Day 7- Plating EBs on gelatin
 15-ml conical tubes
42. Turn on the hood and wipe down with
 Tube rack alcohol.

 Serological pipettes 43. Gather your material and wipe them

down with alcohol before placing them
 Differentiation media, warmed under the hood:

 Retinoic acid (5mM)  Serological pipettes

 Bacteria 10-cm Petri dishes  P100 or p200 pipettors

33. Carefully transfer all the media plus  Wide bore pipette tips
embryoid bodies from each dish into a
separate 15ml conical tube.  24-well tissue culture plates

34. Place the tubes in a rack inside the 37°C  0.1% gelatin in ddH2O
incubator.

52
  Differentiation media, warmed 4. Can you recognize any other cell type in
your plates?
 5mM retinoic acid (RA)
5. Write your observations in your notebook
44. Add 0.5 ml of 0.1% gelatin per well and
and discuss your results.
wait for 5 minutes. Aspirate off the gelatin.
This will gelatin coat your multi-well tissue
culture plate.

45. Add fresh ESC Differentiation media

with 5M RA for the neuron differentiation
and without RA for cardiomyocyte
differentiation to each well. Use 1ml per
well.

46. Using a P100 or P200 pipettor and

special wide bore tips (or 1-ml serological
pipette), gently transfer EBs into the gelatin-
coated plates. Transfer about 5-10 EBs into
each well.

47. Place plates into the 37°C incubator.

Day 9+- Observing the differentiated cells

48. Beating cardiomyocytes and neural cells

with long axonal projections should be
visible under the microscope.

Analysis questions:

1. What percentage of the cells are

differentiated into neurons? How efficient is
the above procedure in directing neuron
differentiation?

2. What percentage of the cells have

developed into beating cardiomyocytes?
How efficient is the above procedure in
directing cells to cardiomyocyte
differentiation?

3. Do you see any neurons in the plates

without RA?

53
Appendix A original culture to a tube containing 900 l
of buffer (10X dilution). In the second step,
Serial Dilution add 100 l of the diluted sample to 9.9 ml
(9900 l) of buffer (100X dilution). In the
Serial dilutions are commonly used in third step, take out 100 l of the later tube
microbiology, chemistry and biotechnology. and add it to a new tube with 9.9 ml of
Serial dilutions are performed to prepare buffer (100X dilution). Our total dilution
solutions that are less concentrated than the factor is 10 x 100 x 100 = 105. So the final
original solution. Serial dilutions are usually solution is 105 times less concentrated than
made in increments of 1000, 100 or 10. A the original solution and it contains 5 x103
small amount of the original solution is cells/ml.
removed and added to the appropriate
amount of buffer. Note, if you were to dilute the original
solution 105 times in only one step, we
For example, if you remove 10 l of your would need to add 100 l of solution to a
original solution and add it to a tube tube containing 9, 999, 900 l (9, 999.9 ml)
containing 990 l of water, you have made a of buffer. This would require a very big
100X dilution. Meaning that your new container that could hold large volumes of
solution is 100 times less concentrated than liquid. It would also be impractical since a
the original solution. If you remove 1l of lot of buffer would be wasted. Therefore,
your diluted solution and add it to a third serial dilution is the best method used for
tube containing 999 l of water, then you large dilutions.
have made another 1000X dilution. So the
concentration of the third tube is 1000 times
less than the second tube and 100,000 (105)
times less than the original tube.

In cell culture techniques, if we are going to

measure a volume of liquid containing cells,
it is more accurate to avoid measuring
amounts less than 50-100 l (there may be
times that we do not have enough cell
solution or not enough buffer solution for
multiple dilution steps, and so we would
Figure 15- 10 5 times dilution of a 5 x 108 cells/ml
have to measure less than 50 l). suspension completed in three steps.

For example, if we have a cell-culture

solution containing 5 x 108 cells/ml and
need to prepare a solution of 5 x 103 cells/ml
(Fig. 15), we need to first add 100 l of the

54
Appendix B 6. A student counts more than 600 cells in
ten squares of a hemocytometer. To get a
more accurate count, he dilutes his cells by
Practice problems
taking out 100 l of cells and mixing them
with 900 l of PBS. He then takes out 50 l
1. A student has a cell suspension of 105
of the diluted sample and mixes it with 50 l
cells/ml. How is he going to prepare 10 ml
of Trypan blue. Using a hemocytometer he
of a 102 cells/ml suspension?
counts 60 cells in ten squares. What is the
concentration of cells in his original culture?
2. A student has a cell suspension of 105
cells/ml. She needs to plate 5 ml of 5000
7. A student is asked to plate 1000 cells per
cells/ml culture into a T25 flask. How is she
well of a 24-well plate with 1ml of media in
going to plate her cells?
each well. He first trypsinizes his cells in a
T25 flask and quenches the trypsin with
3. A student has a cell suspension of 105 media. He then counts the cells using a
cells/ml. She needs to plate 5 ml of 100 hemocytometer. He counts 200 cells in ten
cells/ml culture into a T25 flask. How is she squares. How is he going to plate the cells?
going to plate her cells?

8. A student is asked to plate 100 cells per

well of a 6-well plate using 2 ml of media
4. A student has trypsinized his cells with 1
per well. After trypsinizing she counts 400
ml of trypsin. He then adds 4 ml of media to
cells in 10 squares of a hemocytometer.
quench trypsin. Using a hemocytometer he
How is she going to plate the cells?
counts 150 cells in ten squares. What is the
concentration of his cells? How many total
cells does he have in his culture? 9. A student is asked to plate cells in a T25
at the density of 1000 cells/cm2. She first
counts the cells using a hemocytometer. She
5. A student is interested in counting both
counts 240 cells in five squares. How is she
live and dead cells in a culture. After
going to plate the cells?
collecting the floating cells and pooling with
the trypsinized cells from his culture, he
takes out 50 l and mixes it with 50 l of 10. A student is asked to seed a 12-well
Trypan blue. He then uses a hemocytometer plate with 2000 cells/cm2 using 1 ml of
for counting. He counts 60 dead cells and media per well. Each well of a 12-well plate
300 live cells in five squares. What is the is 3 cm2. He counts 60 cells in ten squares of
concentration of live and dead cells? a hemocytometer. How is he going to plate
the cells?

55
11. A student is asked to freeze his cells
from a T25 flask at the concentration of 106
cells/ml. After trypsinizing his cells with
1ml trypsin, he adds 4 ml of media to
quench the trypsin. Using a hemocytometer,
he counts 315 cells in ten squares. How
much freezing media does he need to freeze
his cells in and how is he going to freeze his
cells?

56
Glossary:
Complete-media- Basal media
Adult stem cells- Stem cells that are found supplemented with additional proteins,
in adult tissue in the body and are able to growth factors and components necessary
regenerate specific types of cells for efficient cell growth.
(multipotent).
Confluency- The percentage of the culture
Anchorage-dependent cells- Cells that vessel’s surface covered by cells.
need to attach to a solid support to survive
and divide. Cytotoxic studies- Studies that involve
measurement of altered metabolism or loss
Anchorage-independent cells- Cells that of viability of cells due to a toxic factor.
can survive without attachment to a solid
support and grow in suspension. Defined media- Basal media plus additional
growth factors, proteins and other
Aseptic techniques- Series of techniques components that are added without the use
and practices used to reduce the chances of of serum. Since the exact components of the
contamination of the cultures by factors added are known, this type of media
microorganisms and to protect laboratory is can be defined.
workers from contamination by cell cultures
and other potentially hazardous material. Density-dependent growth inhibition-
When the cells stop dividing due to
Basal media- Media containing basic insufficient space, nutrients or oxygen
nutrients, vitamins and minerals necessary available to them.
for cells to grow in culture. Basal media
needs to be supplemented with additional Differentiation- A process when cells
growth hormones, growth factors and mature and become specialized.
proteins for efficient cell growth.
Embryoid bodies (EB)- Aggregates of
Bicarbonate buffer- A buffer used in cell embryonic stem cells.
culture media that interacts with CO2 to
keep the pH balanced at about 7.4. Embryonic stem (ES) cells- A type of stem
cell formed very early during development.
Chelator- Molecules that bind to certain ES cells are pluripotent.
metal ions and inactivate the ions so that
they cannot react with other elements. Extracellular matrix- Collection of
molecules outside of cells that provide
Colony- A population of cells that are all structural support for the cells in a tissue in
descendents of a single parental cell. Cells in addition to having other important functions.
a colony are located close together.

57
Feeding- When the old media is removed Micropipettes- Pipettes that are used to
from the culture and replaced with fresh transfer small volumes of liquid between 1-
media. 1000 l.

Filter cube- An optical block with a set of Monolayer- Refers to cells growing in a
filters that allow selection of certain single layer, attached to the bottom of the
wavelengths, used in fluorescence culture vessel.
microscopes.
Mycoplasma- Very small bacteria-like
Fluorophores- Molecules that can absorb microorganisms that can grow rapidly in cell
energy of light at specific wavelengths and cultures without being visible.
emit less energetic fluorescent light.
Passaging- Splitting the cells in a culture
Fusion protein- A protein made by fusion into subcultures.
of two or more different proteins.
Pellet- The components of a mixture that are
IC50- The dosage of a toxic reagent at which precipitated to the bottom after
there is 50% inhibition of colony formation. centrifugation.

Infection- Transfer of viral genetic material Phenol red- A component of the cell culture
into eukaryotic cells. media that is a pH marker and will change
color with the changes in the pH.
Isotonic solutions- Solutions with the same
concentration of solutes as the inside of the Photobleaching- the loss of fluorescent
cells. properties of a fluorophore due to
continuous exposure to light at any
Hayflick’s limit- A phenomenon of normal wavelength.
cells in culture when they divide for a
limited number of times before they stop Plasmids- Small, circular DNA molecules
dividing. that naturally exist in bacteria and some
eukaryotic cells. Plasmids are used as tools
Liquid nitrogen tanks- Tanks filled with in biotechnology to carry foreign DNA
liquid nitrogen, used for long-term storage sequences into cells.
of cells at a very cold temperature.
Plating efficiency (or cloning efficiency)-
Microbial contaminants- Microorganisms The percentage of cells that survive and are
that may grow in cell media and able to divide and form colonies after being
contaminate the culture. plated.

58
Pluripotent- A characteristic of embryonic Supernatant- The liquid component of a
stem cells, which is their ability to generate mixture that stays on top of the pellet after
all cell types. centrifugation.

Propodium Iodide- A fluorescent molecule Transfection- A method used to transfer

that intercalates between nucleic acids (both non-viral recombinant DNA into
RNA and DNA) absorbing light most mammalian cells.
efficiently at 500-550nm and emiting bright
red fluorescent light at 600-650nm Transformed cells- Mammalian cells that
wavelengths. have been immortalized.

Pyrimidines- Cytosine (C), Thymine (T), Transient transfection- Transfection of

and Uracil (U) nucleic acids. cells when the foreign DNA molecule stays
independent of the host genome and is
Pyrimidine dimers- Molecular lesions eventually lost during subsequent cell
formed from adjacent thymine or cytosine divisions.
bases in DNA by formation of covalent
bonds caused by exposure to UV radiation. Trypan blue- A blue dye that is
impermeable to live cells but can go through
Recombinant DNA- A DNA molecule that the porous membranes of dead cells and turn
is made artificially by combining two or them blue.
more different DNA sequences together.
Trypsin- A commonly used dissociating
Self-renewal- A characteristic of stem cells, enzyme which digests the attachment
which is their ability to divide and make proteins on the surface of the cells. Used to
more stem cells. detach the cells from the surface of the
culture vessel.
Serological pipettes- Pipettes that are used
to measure and transfer larger volumes of Turret – Part of a microscope that holds
liquid. Serological pipettes need the help of the objective lenses and can be rotated to put
the “pipette aid” pumps to draw and release an objective lens in the light path.
liquid.
Undefined-media- Basal media plus serum.
Serum- Blood without the cellular Since the exact components of the serum are
components. unknown, this type of media is called
undefined.
Stable transfection- Transfection when the
foreign DNA becomes part of the host
genome and is passed to the next
generations.

59
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524.
Dhara, S. K. and Stice, S. L. (2008) Neural
differentiation of human embryonic stem
cells. J. Cell Biochem. 105(3): 633-640.

Eagle, H. (1959) Amino acid metabolism in

mammalian cell cultures. Science 130: 432.

Freshney, R. I. (2005) Culture of animal

cells: a manual of basic techniques. 5th ed.
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Dulbecco, R. and Freeman, G. (1959)

Plaque formation by the polyoma virus.
Virology 8: 396-397.

Gey, G. O., Coffman, W. D. and Kubicek,

M. T. (1952) Tissue culture studies of the
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Goodsell, D. S. (2001) The Molecular

Perspective: Ultraviolet Light and
Pyrimidine Dimers. The Oncologist 6 (3):
298-299.

Hayflick L and Moorhead, P. S. (1961) The

serial cultivation of human diploid cell
strains. Exp. Cell Res. 25: 585-621.

Verso, M. L. (1971) Some nineteenth-

century pioneers of hematology. Med Hist.
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Moore, G. E. Gerner, R. E. and Franklin H.

A. (1967) Culture of normal human

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