ISG Reporter HEK 293 Cells

Interferon regulatory factor (IRF)-inducible SEAP reporter HEK293 cells

HEK-Blue™ ISG Cells signaling pathway

HEK-Blue™ ISG cells were specifically designed to study the activation of the STING/TBK1/IRF3 signaling pathway by cyclic dinucleotides (CDNs). 

CDNs in the cytosol bind directly to STING leading to TBK1-mediated IRF3 activation and type I interferon (IFN) production [1]. IFNs activate the JAK-STAT pathway with the subsequent activation of IFN-stimulated response elements (ISRE) in the promoters of IFN-stimulated genes (ISG).

More details about STING

Cell line description:

HEK-Blue™ ISG cells were derived from the PEAKrapid cell line (similar to ATCC® CRL-2828™) which itself was derived from the human embryonic kidney (HEK)-293 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.
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™ Solution, 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 but poorly to cytosolic DNA, DMXAA, and canonical CDNs.

Features of HEK-Blue™ ISG cells:

• Fully functional STING/TBK1/IRF signaling pathway
• Readily assessable SEAP reporter activity
• Functionally tested and guaranteed mycoplasma-free

Application of HEK-Blue™ ISG cells:

• Studying the STING/TBK1/IRF signaling pathway

Reference:

1. Wan D. et al., 2020. Research Advances in How the cGAS-STING Pathway Controls the Cellular Inflammatory Response. Front Immunol. 11:615.

Figures

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%).

Specifications

Antibiotic resistance: G418, Zeocin®

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

Quality control:

• Reporter activity is validated upon stimulation with IFN-α or IFN-β and IRF3 activators.
• These cells are guaranteed mycoplasma-free.

Contents

• 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).

Shipped on dry ice (Europe, USA, Canada and some areas in Asia)

Details

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 [1, 2], and acts as a direct sensor of cyclic dinucleotides (CDNs) [3]. 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 [4]. CDNs bind directly to and activate STING leading to TANK Binding Kinase 1 (TBK1)-dependent interferon regulatory factor 3 (IRF3) activation and type I IFN production. IFNs then activate the JAK-STAT pathway with subsequent activation of IFN-stimulated response elements (ISRE) in the promoters of IFN-stimulated genes (ISG).

1. Wan D. et al., 2020. Research Advances in How the cGAS-STING Pathway Controls the Cellular Inflammatory Response. Front Immunol. 11:615.
2. Ishikawa H. & Barber, G., 2008. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455, 674–678.
3. Burdette D.L. et al., 2011. STING is a direct innate immune sensor of cyclic di-GMP. Nature 478(7370):515-8.
4. 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.

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