293-Dual™ hSTING-R232 Cells
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Dual IRF and IFN-β reporter 293 cells expressing R232 isoform of human STING
3-7 x 10e6 cells
293-Dual™ hSTING-R232 Cells (ISG-SEAP/KI-[IFN-β]Lucia)
293-Dual™ hSTING-R232 (ISG/KI-IFNb) cells were generated from 293-Dual™ Null (ISG/KI-IFNb) cells by stable transfection of the R232 isoform of human STING (hSTING).
Genomic studies indicate that this isoform, which contains an arginine at position 232 (R232), is the most prevalent with an isoform occurrence of ~60% in the human population [1, 2]. This isoform is preferentially activated by 2’5’linkage-containing cGAMP isomers .
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. Jin L. et al., 2011. Identification and characterization of a loss-of-function human MPYS variant. Genes Immun. 12(4):263-9.
3. Zhang X. et al., 2013. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol. Cell. 51:226–235.
Dose-responses of 293-Dual™ hSTING R232 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™ hSTINGR232 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,Rp) (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™ hSTING-R232 (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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