HEK-Blue™ ISG-KO-STING Cells
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HEK293 - STING Knockout IRF-reporter cells
3-7 x 10e6 cells
STING Knockout IRF Reporter Cell Line
HEK-Blue™ ISG-KO-STING cells were specifically designed to study the STING/TBK1/IRF3 signaling pathway. HEK-Blue™ ISG-KO-STING cells were generated from the HEK-Blue™ ISG cells through the stable knockout of the STING gene.
The parental HEK-Blue™ ISG cells were derived from the PEAKrapid cell line (similar to ATCC® CRL-2828™) which itself was derived from the HEK293 cell line. Unlike their parental cell line, HEK-Blue™ ISG-KO-STING cells do not respond to cytosolic DNA, CDNs and xanthenone derivatives, such as DMXAA.
HEK-Blue™ ISG-KO-STING cells can detect bioactive type I IFNs through the activation of the JAK-STAT-IRF9 pathway.
HEK-Blue™ ISG-KO-STING cells express a secreted embryonic alkaline phosphatase (SEAP) under the control of the IRF-inducible promoter comprised of five IFN-stimulated response elements (ISRE) fused to an ISG54 minimal promoter.
Levels of SEAP in the supernatant can be easily determined with QUANTI- Blue™, a reagent that turns purple/blue in the presence of SEAP and by reading the OD at 620-655 nm.
This product is covered by a Limited Use License (See Terms and Conditions).
1. Ishikawa H. & Barber G., 2008. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455, 674–678.
2. Burdette DL. et al., 2011. STING is a direct innate immune sensor of cyclic di-GMP. Nature 478(7370):515-8.
3. ;Wu J. et al., 2013. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339(6121):826-30.
Stimulation of HEK-Blue™ ISG-KO-STING and HEK-Blue™ ISG cells (wild-type cell line) with poly(dA:dT)/LyoVec™ (1 µg/ml), 2’3’-cGAMP (1 µg/ml), 3’3’-cGAMP (10 µg/ml) and 2’3’-c-di-AMP (10 µg/ml). Human IFN-α (1x103 U/ml) and IFN-β (1x103 U/ml) serve as positive controls. After 24h incubation, IRF activation was determined using QUANTI-Blue™, a SEAP detection reagent, and by reading the optical density (OD) at 655 nm. The IRF induction of each ligand is expressed relative to that of mIFN-β at 1x103 U/ml (taken as 100%).
Stimulation of HEK-Blue™ ISG-KO-STING and HEK-Blue™ ISG cells (wild-type cell line) with poly(dA:dT)/LyoVec™ (1 µg/ml), 2’3’-cGAMP (1 µg/ml), 3’3’-cGAMP (10 µg/ml) and 2’3’-c-di-AMP (10 µg/ml). Human IFN-α (1x103 U/ml) and IFN-β (1x103 U/ml) serve as positive controls. After 24h incubation, IRF activation was determined using QUANTI-Blue™, a SEAP detection reagent, and by reading the optical density (OD) at 655 nm. The difference in activity between the two cell lines is expressed as the ratio WT/KO, which was obtained by dividing each value in figure 1A for HEK-Blue™ ISG (WT) cells by the corresponding value for HEK-Blue™ ISG-KO-STING cells.
Quality control: STING knockout is verified by functional assays (see validation sheet) and DNA sequencing. These cells are guaranteed mycoplasma-free.
Growth Medium: DMEM, 4.5 g/l glucose, 10% (v/v) fetal bovine serum (FBS), 50 U/ml penicillin, 50 mg/ml streptomycin, 100 µg/ml Normocin™, 2 mM L-glutamine supplemented with Zeocin™ selective antibiotic only
Test Medium: DMEM, 4.5 g/l glucose, 10% (v/v) heat-inactivated FBS (30 min at 56°C), 50 U/ml penicillin, 50 µg/ml streptomycin, 100 µg/ml Normocin™, 2 mM L-glutamine
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- 1 vial containing 3-7 x 106 cells
- 100 μl Zeocin™ (100 mg/ml)
- 1 ml Normocin™ (50 mg/ml)
- 1 pouch of QUANTI-Blue™ (SEAP detection medium)
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STING (stimulator of interferon genes; also known as TMEM173, MITA, MPYS and ERIS) is essential for the interferon (IFN) response to cytosolic nucleic acids, such as microbial or self-DNA , and acts as a direct sensor of cyclic dinucleotides (CDNs) .
CDNs are important second messenger molecules in bacteria, affecting numerous responses of the prokaryotic cell. In mammalian cells, CDNs act as agonists of the innate immune response . CDNs bind directly to and activate STING leading to a potent type I interferon (IFN) response.Back to the top