293-Dual™ hSTING-A162 Cells

293-Dual™ hSTING-A162 Cells (ISG-SEAP/KI-[IFN-β]Lucia)

293-Dual™ hSTING-A162 (ISG/KI-IFNb) cells were generated from 293-Dual™ Null (ISG/KI-IFNb) cells by stable transfection of the A162 isoform of human STING (S162A).

The allele A162 contains a unique point mutation (S162A) placed at the cyclic-dinucleotide-binding site which confers sensitivity to DMXAA, a potent tumor vascular disrupting agent in mice [2].

In the absence of this mutation, DMXAA has no effect on human STING [3].

293-Dual™ hSTING-A162 (ISG/KI-IFNb) cells are resistant to blasticidin, hygromycin, and Zeocin® .

References:

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. Gao P. et al., 2013. Structure-function analysis of STING activation by c[G(2’,5’)pA(3’,5’)p] and targeting by antiviral DMXAA. Cell 154(4):748-62.
3. 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.

Figures

Dose-responses of 293-Dual™ hSTING A162 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 A162 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).

Specifications

Antibiotic resistance: blasticidin, hygromycin, and Zeocin®

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™

Quality Control:

• 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.

Guaranteed mycoplasma-free.

Contents

• 3-7 x 10⁶ of 293-Dual™ hSTING-A162 (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)

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

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