293-Dual™ mSTING Cells
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Dual IRF and IFN-β reporter 293 cells expressing murine STING
3-7 x 10e6 cells
293-Dual™ mSTING Cells (ISG-SEAP/KI-[IFN-β]Lucia)
293-Dual™ mSTING (ISG/KI-IFNb) cells were generated from 293-Dual™ Null (ISG/KI-IFNb) cells by stable transfection of the murine STING gene.
These cells exhibit a robust response to STING agonists such as 2’3’-cGAMP and 3’3’-cGAMP. In addition, unlike cells with human STING, these cells respond to DMXAA (5,6-Dimethylxanthenone 4-acetic acid) which induces a potent IFN response by activating murine STING [2, 3].
1. Yi G. et al., 2013. Single nucleotide polymorphisms of human STING can affect innate immune response to cyclic dinucleotides. PLoS One. 8(10):e77846.
2. Conlon J. et al., 2013. Mouse, but not human STING, binds and signals in response to the vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid. J Immunol 190(10):5216-25.
3. Zhang H. et al., 2015. Rat and human STINGs profile similarly towards anticancer/antiviral compounds. Sci Rep. 5:18035.
Dose-responses of 293-Dual™ mSTING cells stimulated with 2’3’-cGAMP, 3’3’-cGAMP and DMXAA. After 24h incubation, IRF induction was assessed by measuring the levels of SEAP using QUANTI‑Blue™ and by reading the optical density (OD) at 655 nm.
293-Dual™ mSTING cells were stimulated with 3’3’-cGAMP (30 μg/ml), 3’3’-cGAMP fluorinated (10 μg/ml), 2’3’-cGAMP (30 μg/ml,) 2’3’-cGAM(PS)2 (Rp,Sp) (10 μg/ml), DMXAA (30 μg/ml) and human IFN-α (30 IU/ml). After 24h incubation, IFN-β induction was assessed by measuring the levels of Lucia luciferase using QUANTI-Luc™ and by reading the relative light units (RLUs) in a luminometer. The IFN-β response is expressed as a fold induction (calculated by dividing the RLUs for the treated cells by the RLUs for the untreated cells).
Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™
- Reporter activity has been validated by stimulating the cells with human interferon-β (hIFN-β) and IRF3 activators, such as c-di-AMP and cGAMP.
- The biallelic replacement of the hIFN-β coding sequence with the Lucia luciferase open reading frame (ORF) has been verified by PCR and sequencing.
- The inability to produce IFN-β has been confirmed by ELISA.
- The cell line stability for 20 passages following thawing has been verified.
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- 3-7 x 106 of 293-Dual™ mSTING (ISG/KI-IFNb) cells in a cryovial or shipping flask
- 1 ml of Blasticidin (10 mg/ml)
- 1 ml of Hygromycin (100 mg/ml)
- 1 ml of Zeocin® (100 mg/ml)
- 1 ml of 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)
- 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)
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