HEK-Blue™ IL-4/IL-13 Cells - InvivoGen

STAT6 SEAP
JAK1 TyK2
IL-13
IL-4
STAT6
IL-13Rα1
STAT6
STAT6
IL-4Rα
P P
STAT6
HEK-Blue IL-4/IL-13 Cells
Interleukin-4 and interleukin-13 sensor cells
Catalog # hkb-stat6
For research use only
Version # 12C15-MM
pRODUCT INfORmaTION
Contents:
• 1 vial of HEK-Blue IL4/IL-13 Cells (5-7 x 10 6 cells)
• 100 µl Blasticidin (10 mg/ml). Store Blasticidin at 4˚C for 6 months
or at -20˚C for 1 year.
• 100 µl Zeocin (100 mg/ml). Store Zeocin at 4˚C for 6 months or
at -20˚C for 1 year.
• 1 ml Normocin (50 mg/ml). Normocin is a formulation of three
antibiotics active against mycoplasmas, bacteria and fungi. Store
at -20°C. Product is stable for 18 months when stored at -20°C.
• 1 pouch of QUANTI-Blue (SEAP detection medium). Store
QUANTI-Blue pouch at room temperature for up to 6 months.
Reconstituted QUANTI-Blue medium is stable 2 weeks at 4˚C.
Keep reconstituted QUANTI-Blue away from light.
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.
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 IL4/IL-13 cells should not be passaged more than 20
times to remain fully efficient. HEK-Blue IL4/IL-13 cells should be
maintained in Growth Medium supplemented with the two selective
antibiotics, Zeocin (100 mg/ml) and Blasticidin (10 µg/ml).
Antibiotic pressure with Blasticidin is required to maintain the
plasmid coding for human STAT6 gene and Zeocin is required to
maintain the plasmid coding for SEAP.
INTRODUCTION
The transcription factor STAT6 is
activated primarily by two cytokines
with overlapping biologic functions,
IL-4 and IL-13. It can also be
activated by IFN-a in a cell-specific
manner. In non-hematopoietic
cells, IL-4 and IL-13 bind a
receptor complex composed of
the IL-4Ralpha and IL-13Ralpha1.
Upon ligand binding, the receptor
complex activates the receptorassociated
Janus kinases (JAK1 and
Tyk2) leading to the recruitment of
STAT6 and its phosphorylation.
Activated STAT6 forms homodimers
that translocate to the nucleus where
they bind the promoter of responsive
genes inducing gene transcription.
IL-4
IL-13
TyK2
IL-4Rα
IL-13Rα1
JAK1
P P
STAT6
STAT6
STAT6
SEAP
Cell lINe DesCRIpTION
HEK-Blue IL-4/IL-13 Cells are specifically designed to monitor
the activation of the STAT6 pathway induced by IL-4 and IL-13
(and IFNa). These cells were generated by stably introducing the
human STAT6 gene into HEK293 cells to obtain a fully active
STAT6 signaling pathway. The other genes of the pathway are
naturally expressed in sufficient amounts. Furthermore,
HEK-Blue IL-4/IL-13 Cells stably express the reporter gene
secreted embryonic alkaline phosphatase (SEAP) under the control
of the IFNb minimal promoter fused to four STAT6 binding sites.
Activation of the STAT6 pathway in HEK-Blue IL-4/IL-13 Cells
induces the expression of the reporter gene. SEAP which is
secreted in the supernatant is easily detectable when using
QUANTI-Blue a medium that turns purple/blue in the presence of
SEAP.
Quality control:
Expression of STAT6 was confirmed by RT-PCR. HEK-Blue
IL-4/IL-13 Cells were stimulated by various cytokines. Production of
SEAP was detected following stimulation by IL-4, IL13 and and also
type I IFNs (essentially IFN-a) but to a lesser extent. These cells are
guaranteed mycoplasma-free.
STAT6
STAT6
TECHNICAL SUPPORT
Toll free (US): 888-457-5873
Outside US: (+1) 858-457-5873
Europe: +33 562-71-69-39
E-mail: info@invivogen.com
Website: www.invivogen.com
3950 Sorrento Valley Blvd. Suite 100
San Diego, CA 92121 - USA

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 with 20% fetal bovine serum and 10%
(v/v) DMSO
• Test Medium: DMEM, 4.5 g/l glucose, 10% (v/v) heat-inactivated
fetal bovine serum (30 min at 56ºC), 50 U/ml penicillin, 50 mg/ml
streptomycin, 100 mg/ml Normocin , 2 mM L-glutamine
Note: Heat-inactivated FBS is also commercially available.
frozen cells
1- Thaw the vial by gentle agitation in a 37°C 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% ethanol.
Note: All of the operations 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 T-25 tissue culture flask containing 5 ml
of growth medium without selective antibiotics.
7- Place the culture at 37˚C in 5% CO2.
Day 2:
- Prepare QUANTI-Blue following the instructions on the pouch.
- Add 180 ml of resuspended QUANTI-Blue per well of a
flat-bottom 96-well plate.
- Add 20 ml of induced HEK-Blue IL-4/IL-13 Cells supernatant.
- Incubate the plate at 37°C incubator for 1-3 h.
- Determine SEAP levels using a spectrophotometer at 620-655 nm.
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 info@invivogen.com
RelaTeD pRODUCTs
product
Blasticidin (100 mg)
Zeocin (1 g)
Normocin
QUANTI-Blue (5 pouches)
Catalog Code
ant-bl-1
ant-zn-1
ant-nr-1
rep-qb1
Cell maintenance
- Maintain and subculture the cells in growth medium supplemented
with 10 µg/ml of blasticidin and 100 µg/ml of Zeocin .
- Renew growth medium 2 times a week.
- Cells should be passaged when a 70-80% confluency is reached. Do
not let the cell grow to 100% confluency.
Il-4/Il-13 Detection
Day 1:
- Add 20 ml of each sample per well of a flat-bottom 96-well plate.
- Add 20 ml of IL-4 or IL-13 at 10 ng/ml (positive control) in one well.
- Add 20 ml of TNF-a at 100 ng/ml (negative control, other cytokines
can be used) in one well.
- Prepare a cell suspension of HEK-Blue IL-4/IL-13 Cells
at ~280,000 cells per ml in test medium (containing 10% v/v
heat-inactivated FBS).
Note: Some FBS may contain alkaline phosphatases that can interfere
with SEAP quantification.We recommend to use heat-inactivated FBS
to inactivate these enzymes which are thermosensitive.
- Add 180 ml of cell suspension (~50,000 cells) per well.
- Incubate the plate at 37°C in a CO2 incubator for 20-24 h.
TECHNICAL SUPPORT
Toll free (US): 888-457-5873
Outside US: (+1) 858-457-5873
Europe: +33 562-71-69-39
E-mail: info@invivogen.com
Website: www.invivogen.com
3950 Sorrento Valley Blvd. Suite 100
San Diego, CA 92121 - USA