Hek Blue Htlr7 Tds

HEK-Blue hTLR7 Cells
SEAP Reporter 293 cells expressing the human TLR7 gene

Catalog # hkb-htlr7
For research use only

Version # 12I12-MM
PRODUCT iNFORMATiON
BACKGROUND
Contents and Storage
TLR7, which is abundantly expressed in lung, placenta, spleen and PBL,

is phylogenetically close to TLR8 and TLR91. TLR7 recognizes small
synthetic molecules such as loxoribine and R848, an imidazoquinoline
compound2. TLR7 signaling involves the MyD88-dependent signaling
cascade and induces the production of IFN-, TNF- and IL-12.
Sequence-specific single-stranded RNA (ssRNA) was identified as the
natural ligand of TLR73, 4. ssRNA sequences derived from HIV-1 or the
influenza virus were shown to induce the production of proinflammatory
cytokines in PDC. Furthermore, it has been demonstrated that TLR7 is
involved in sequence-specific sensing of ssRNA in human macrophages5.
TLR7 signaling is abrogated by chloroquine indicating that it is dependent
on endosomal acidification.
1 vial of HEK-Blue hTLR7 Cells (5-7 x 106 cells) in Freezing Medium

IMPORTANT: Cells are shipped frozen. If cells are not frozen upon
arrival, contact InvivoGen immediately.
100 l Blasticidin selective antibiotic (10 mg/ml). Store at -20C.*
100 l Zeocin selective antibiotic (100 mg/ml). Store at -20C.*
* Selective antibiotics are stable for at least 1 year when stored at -20C.
1 ml Normocin (50 mg/ml). Normocin is a formulation of three
antibiotics active against mycoplasmas, bacteria and fungi. Store at -20C.
Product is stable for 18 months when stored at -20C.
1 pouch of HEK-Blue Detection, a cell culture medium (50 ml) for
real-time detection of SEAP. Store pouch at room temperature. Store
reconstituted HEK-Blue Detection at 4C for 2 weeks.
PRODUCT DESCRiPTiON
HEK-Blue hTLR7 cells are designed for studying the stimulation of
human TLR7 (hTLR7) by monitoring the activation of NF-kB.
HEK-Blue hTLR7 cells were obtained by co-transfection of the hTLR7
gene and an optimized secreted embryonic alkaline phosphatase (SEAP)
reporter gene into HEK293 cells. The SEAP reporter gene is placed under
the control of the IFN-b minimal promoter fused to five NF-kB and
AP-1-binding sites. Stimulation with a TLR7 ligand activates NF-kB and
AP-1 which induce the production of SEAP.
Levels of SEAP can be easily determined with HEK-Blue Detection, a
cell culture medium that allows for real-time detection of SEAP.
HEK-Blue Detection is a one-step procedure and extremely simple to
use. It is applicable to high-throughput screening. HEK-Blue Detection
contains all the nutrients necessary for cell growth and a specific SEAP
color substrate. The hydrolysis of the substrate by SEAP produces a
purple/blue color that can be easily detected with the naked eye or
measured with a spectrophotometer.
SEAP activity can also be assessed using the alkaline phosphatase detection
medium, QUANTI-Blue. Following cell activation, the two-step
QUANTI-Blue assay detects SEAP in the cell supernatant. This
colorimetric assay allows the same cell cultures to be repeatedly sampled
for kinetic studies or further experimention.
Note: With the QUANTI-Blue assay, stimulate HEK-Blue hTLR7 cells
in a culture medium containing heat-inactivated fetal bovine serum
(FBS), as some FBS may contain alkaline phosphatases that can interfere
with SEAP quantification.
HEK293 cells express endogenous levels of TLR3, TLR5 and NOD1.

Note: The appropriate parental cell line for HEK-Blue hTLR7 cells is
HEK-Blue Null1-k cells (SEAP reporter cells which do not express hTLR7).
TECHNICAL SUPPORT
Toll free (US): 888-457-5873
Outside US: (+1) 858-457-5873
Europe: +33 562-71-69-39
E-mail: [email protected]
Website:www.invivogen.com
1. Chuang TH. & RJ. Ulevitch, 2000. Cloning and characterization of a sub-family of human
toll-like receptors: hTLR7, hTLR8 and hTLR9. Eur Cytokine Netw, 11:372-8. 2. Hemmi H. et
al., 2002. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent
signaling pathway. Nat Immunol, 3:196-200. 3. Heil F. et al., 2004. Species-specific recognition
of single-stranded RNA via toll-like receptor 7 and 8. Science. 303:1526-9. 4. Diebold SS. et al.,
2004. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded
RNA. Science. 303:1529-31. 5. Gantier MP. et al., 2008. TLR7 is involved in sequence-specific
sensing of single-stranded RNAs in human macrophages. J immunol. 180; 2117-24.
Handling Cells Upon Arrival

We strongly recommend that you propagate the cells, using the provided
procedure, as soon as possible. This will ensure the best cell viability and
assay performance. Frozen cells may be placed in liquid nitrogen until you are
ready to thaw and propagate them, however, this may reduce cell viability.
Product Warranty
InvivoGen warrants that cells shall be viable upon shipment from
InvivoGen for a period of thirty days, provided they have been properly
stored and handled during this period.
Quality Control
Expression of the human TLR7 gene was confirmed by RT-PCR.
HEK-Blue hTLR7 Cells were stimulated by TLR7 agonists. As
expected, TLR7 agonists induced the production of SEAP. These cells are
guaranteed mycoplasma-free.
Cell Line Stability

Cells will undergo genotypic changes resulting in reduced responsiveness
over time in normal cell culture conditions. Genetic instability is a biological
phenomenon that occurs in all stably transfected cells. Therefore, it is critical
to prepare an adequate number of frozen stocks at early passages.
HEK-Blue hTLR7 Cells should not be passaged more than 20 times to
remain fully efficient. HEK-Blue hTLR7 Cells should be maintained in
Growth Medium as described below in the presence of
Normocin (100 g/ml) and the two selective antibiotics, Blasticidin (10
g/ml) and Zeocin (100 g/ml). Antibiotic pressure with Blasticidin is
required to maintain the plasmid coding for hTLR7, and Zeocin is required
to maintain the plasmid coding for SEAP.
3950 Sorrento Valley Blvd. Suite 100

San Diego, CA92121 - USA
USE RESTRiCTiONS
These cells are distributed for research purposes only.
This product is covered by a Limited Use License. By use of this product,
the buyer agrees the terms and conditions of all applicable Limited Use
Label Licenses. For non-research use, such as screening, quality control or
clinical development, contact [email protected]
SAFETy CONSiDERATiONS
Biosafety Level 2
HANDLiNG PROCEDURES
Required Cell Culture Medium
Growth Medium: DMEM, 4.5 g/l glucose, 10% (v/v) fetal bovine serum, 50
U/ml penicillin, 50 mg/ml streptomycin, 100 mg/ml Normocin, 2 mM
L-glutamine
Freezing Medium: DMEM, 4.5 g/l glucose, 20% (v/v) fetal bovine
serum, 10% (v/v) DMSO
initial Culture Procedure

The first propagation of cells should be for generating stocks for future
use. This ensures the stability and performance of the cells for subsequent
experiments.
1- Thaw the vial by gentle agitation in a 37C water bath. To reduce the
possibility of contamination, keep the O-ring and cap out of the water.
Thawing should be rapid.
2- Remove the vial from the water bath as soon as the contents are thawed, and
decontaminate by dipping in or spraying with 70% (v/v) ethanol.
Note: All steps from this point should be carried out under strict aseptic
conditions.
3- Transfer cells in a larger vial containing 15 ml of pre-warmed Growth
Medium. Do not add selective antibiotics until the cells have been
passaged twice.
4- Centrifuge vial at 1000-1200 RPM (RCF 200-300 g) for 5 minutes.
5- Remove supernatant containing the cryoprotective agent and resuspend
cells with 1 ml of growth medium without selective antibiotics.
6- Transfer the vial contents to a 25 cm2 tissue culture flask
containing 5 ml of growth medium without selective antibiotics.
7- Place the culture at 37C in 5% CO2.
Frozen Stock Preparation

1- Resuspend cells at a density of 5-7 x 106 cells/ml in freezing medium
freshly prepared with cold growth medium.
Note: A T-75 culture flask typically yields enough cells for preparing 3-4
frozen vials.
2- Aliquot 1 ml cells into cryogenic vials.
3- Place vials in a freezing container (Nalgene) and store at -80C
overnight.
4- Transfer vials to liquid nitrogen for long term storage.
Note: If properly stored, cells should remain stable for years.
Cell maintenance
1- Maintain and subculture the cells in growth medium supplemented
with 10 g/ml of Blasticidin and 100 g/ml of Zeocin.
2- Renew growth medium 2 times a week.
3- Cells should be passaged when a 70-80% confluency is reached, detach
the cells in presence of PBS by tapping the flask or by using a cell scraper.
Do not let the cells grow to 100% confluency.
Note: The response of HEK-Blue hTLR7 Cells can be altered by the
action of trypsin. Do not use trypsin to detach HEK-Blue hTLR7 Cells.
TECHNICAL SUPPORT
Toll free (US): 888-457-5873
Outside US: (+1) 858-457-5873
Europe: +33 562-71-69-39
E-mail: [email protected]
Website:www.invivogen.com
TLR7 Stimulation determined using HEK-Blue Detection

HEK-Blue Detection is a cell culture medium that allows the detection
of SEAP as the reporter protein is secreted by the cells.
Note: Before the test, the cells should be 50-80% confluent.
1- Add 20 ml of each sample per well of a flat-bottom 96-well plate.
2- Add 20 ml of a positive control (such as CL264, 5 mg/ml) in one well.
3- Add 20 ml of a negative control (such as sterile, endotoxin-free water)
in one well.
4- Remove HEK-Blue hTLR7 Cells from the incubator and discard
growth medium.
5- Gently rinse cells with pre-warmed 5-10 ml PBS (for a T-75 flask).
6- Add 2-5 ml pre-warmed PBS (for a T-75 flask) and place the cells
at 37C for 1- 2 min, detach the cells by tapping the flask or by using a
cell scraper. Dissociate cell clumps by gently pipetting up and down.
Note: Do not use trypsin to detach HEK-Blue hTLR7 Cells.
7- Count cells which have been resuspended in pre-warmed PBS.
Note: Avoid centrifugation of HEK-Blue hTLR7 Cells.
8- Prepare a cell suspension ~220,000 cells per ml in HEK-Blue
Detection medium and immediately add 180 ml of the cell
suspension (~40,000 cells) per well.
Note: Avoid prolonged incubation of cells at room temperature in
HEK-Blue Detection medium as it may lead to high background or false
positive readings.
9- Incubate the plate at 37C in 5% CO2 for 6-16 h. SEAP can be
observed with naked eye and determined using a spectrophotometer
at 620-655 nm.
Note:
- Strong TLR induction in HEK-Blue Detection medium may result in
cell cytoxicity, if this is detrimental to your experiment, we recommend the
use of the two-step QUANTI-Blue assay.
- With the QUANTI-Blue assay, stimulate HEK-Blue hTLR7 Cells in a
culture medium containing heat-inactivated fetal bovine serum (FBS; 30
min at 56C), as some FBS may contain alkaline phosphatases that can
interfere with SEAP quantification. Heat-inactivated FBS is also
commercially available.
Specificity of HEK-Blue hTLR7 Cells

As HEK293 cells express endogenous levels of TLR3, TLR5 and NOD1,
HEK-Blue hTLR7 Cells will respond to their cognate ligands, such as
poly(I:C), flagellin and iE-DAP, respectively. In order to identify TLR7specific responses, we recommend to use HEK-Blue Null1-k Cells as a
control cell line.
Note: HEK-Blue hTLR7 Cells may be stimulated in a TLR7-independent
manner as NF-kB/AP-1 can be activated by a wide variety of stimuli (e.g.
TNF-a and PMA).
RELATED PRODUCTS
Product
HEK-Blue Null1-k Cells
Blasticidin (100 mg)

Zeocin (1 g)
Normocin
HEK-Blue Detection
QUANTI-Blue
CL264 (TLR7 ligand)
CL264 Biotin (labeled TLR7 ligand)
CL264 FITC (labeled TLR7 ligand)
Catalog Code
hkb-null1k
ant-bl-1
ant-zn-1
ant-nr-1
hb-det2
rep-qb1
tlrl-c264
tlrl-bc264
tlrl-fc264
3950 Sorrento Valley Blvd. Suite 100

San Diego, CA92121 - USA

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HEK-Blue hTLR7 Cells

SEAP Reporter 293 cells expressing the human TLR7 gene

For research use only

Contents and Storage

TLR7, which is abundantly expressed in lung, placenta, spleen and PBL,

1 vial of HEK-Blue hTLR7 Cells (5-7 x 106 cells) in Freezing Medium

HEK293 cells express endogenous levels of TLR3, TLR5 and NOD1.

Handling Cells Upon Arrival

Cell Line Stability

3950 Sorrento Valley Blvd. Suite 100

initial Culture Procedure

Frozen Stock Preparation

TLR7 Stimulation determined using HEK-Blue Detection

Specificity of HEK-Blue hTLR7 Cells

Blasticidin (100 mg)

3950 Sorrento Valley Blvd. Suite 100

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