293-Dual™ hSTING-H232 Cells
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Dual IRF and IFN-β reporter 293 cells expressing H232 isoform of human STING (R232H)
3-7 x 10e6 cells
293-Dual™ hSTING-H232 Cells (ISG-SEAP/KI-[IFN-b]Lucia)
293-Dual™ hSTING-H232 (ISG/KI-IFNb) cells were generated from 293-Dual™ Null (ISG/KI-IFNb) cells by stable transfection of the H232 isoform of human STING (hSTING). R232H has been identified as a natural variant allele of STING occurring in ~14% of the human population .
The H232 isoform contains a single amino acid substitution R232H. This isoform has a diminished response to bacterial and metazoan CDNs when compared to the prevalent R232 hSTING allele [1, 2].
H232 has been the most commonly used hSTING variant in published structural studies.
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. Diner E. et al., 2013. The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING. Cell Rep 3(5):1355-61.
Dose-responses of 293-Dual™ hSTING H232 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™ hSTING H232 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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- 1 vial of 293-Dual™ hSTING-H232 (ISG/KI-IFNb) Cells (3-7 x 106 cells)
- 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 pouch of QUANTI-Luc™ (Lucia luciferase detection medium)
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