ISG Reporter HEK 293 Cells
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Interferon Regulatory Factor (IRF)-Inducible SEAP Reporter HEK293 Cells
3-7 x 10e6 cells
Interferon regulatory factor (IRF)-inducible SEAP reporter HEK293 cells
HEK-Blue™ ISG cells were specifically designed to study the activation of the STING/TBK1/IRF3 signaling pathway by CDNs.
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.
HEK-Blue™ ISG 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.
CDNs in the cytosol bind directly to STING leading to TBK1-mediated IRF3 activation and type I IFN production. IFNs activate the JAK-STAT pathway with the subsequent activation of IFN-stimulated response elements (ISRE) in the promoters of IFN-stimulated genes (ISG). Hence, the presence of CDNs in the cytosol of HEK-Blue™ ISG cells will induce the production of the IRF-inducible SEAP reporter directly by activating the STING/TBK1/IRF3 pathway and indirectly through the activation of the JAK/STAT/IRF9 pathway with type I IFN.
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.
HEK-Blue™ ISG cells respond strongly to non-canonical CDNs, namely 2’3’-cGAMP and 2’2’-cGAMP but poorly to cytosolic DNA, DMXAA and canonical cyclic dinucleotides (CDNs).
Response of HEK-Blue™ ISG cells to various CDNs, cytosolic dsDNA and IFN-β. HEK-Blue™ ISG cells were stimulated with 1x103 U/ml human IFN-β, 1 μg/ml poly(dA:dT)/LyoVec™, 30 μg/ml 2’3’-cGAMP, 3’3’-cGAMP, 3’3’-cGAMP Fluorinated, 2’3’-c-di-AMP, cAIMP, and 2’3’-c-di-AM(PS)2 (Rp,Rp). 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 as % activity relative to that of human IFN-β at 1x103 U/ml (taken as 100%).
Growth Medium: DMEM, 4.5 g/l glucose, 10% (v/v) fetal bovine serum (FBS), Pen-Strep (100 U/ml - 100 µg/ml), 100 µg/ml Normocin™, 2 mM L-glutamine
Test Medium: DMEM, 4.5 g/l glucose, 10% (v/v) heat-inactivated FBS (30 min at 56°C), Pen-Strep (100 U/ml - 100 µg/ml), 100 µg/ml Normocin™, 2 mM L-glutamine
- Reporter activity is validated upon stimulation with IFN-α or IFN-β and IRF3 activators such as 2’2’-cGAMP.
- These cells are guaranteed mycoplasma-free.
- 1 vial containing 3-7 x 106 cells
- 1 ml Zeocin™ (100 mg/ml)
- 1 ml Normocin™ (50 mg/ml)
- 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent).
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