B16-Blue™ ISG-KO-STING Cells

STING Knockout IRF-Inducible SEAP Reporter B16 Melanocytes

B16-Blue™ ISG-KO-STING cells can be used for the study of the STING signaling pathway and for the detection of bioactive murine types I and II IFNs by monitoring the activation of the JAK/STAT pathway.

B16-Blue™ ISG-KO-STING cells were generated from the B16-Blue™ ISG cell line, a murine B16- F1 melanoma-derived cell line, through stable knockout of the STING gene. They express the secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of the I-ISG54 promoter which is comprised of the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE.

B16-Blue™ ISG-KO-STING cells do not respond to cytosolic DNA, DMXAA, canonical and non-canonical CDNs while retaining the ability to respond to type I and type II IFNs.

Stimulation of these cells with IFN triggers the activation of the I-ISG54 promoter and the production of SEAP. Levels of SEAP in the supernatant can be easily determined using QUANTI- Blue™, a reagent that turns purple/blue in the presence of SEAP, by reading the OD at 620-655 nm.

B16-Blue™ ISG-KO-STING cells are resistant to Zeocin®.

Figures

Analysis of lysates from the B16-Blue™ (WT) and B16-Blue™ KO-STING (KO) cells using Anti-STING, followed by an HRP-conjugated anti-rabbit secondary antibody. The arrow indicates the expected band for the STING protein (43 KDa).

Response of B16-Blue ISG and B16-Blue ISG-KO-STING to CDNs and IFN-β:
Cells were stimulated with 30 µg/ml of the cyclic dinucleotides, and 10³  U/ml of mIFN-β. Cells were not permeabilized.
After 24h incubation, the levels of IRF-induced SEAP were determined using QUANTI-Blue™.

Specifications

Murine IFN Detection range
• mIFN-α: 10e2 - 10e4 IU/ml
• mIFN-β: 10e2 - 10e4 IU/ml
• mIFN-γ: 0.1 ng - 1 µg/ml

Antibiotic resistance: Zeocin®

Quality control:

• STING knockout is verified by functional assays and DNA sequencing to confirm frameshift mutation/deletion
• These cells are guaranteed mycoplasma-free

Growth medium: DMEM, 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 µg/ml Normocin™, 100 U/ml penicillin, 100 µg/ml streptomycin

Contents

• 1 vial containing 3-7 x 10⁶ cells
• 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)

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

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