B16-Blue™ ISG Cells

Interferon Regulatory Factor-Inducible SEAP Reporter B16 Melanocytes

B16-Blue™ ISG cells allow the detection of bioactive murine type I and II IFNs by monitoring the activation of the JAK/STAT pathway.

B16-Blue™ ISG cells can also be used study the activation of the TBK1/IRF3 pathway by cytosolic DNA, dsRNA or CDNs.

B16-Blue™ ISG cells were derived from the murine B16 F1 melanoma cell line. 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.

Stimulation of B16- Blue™ ISG cells with IFNs, CDNs, such as cGAMP, or type I IFN inducers, such as transfected poly(dA:dT), 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 cells are resistant to Zeocin®.

Figures

RIG-I activation with 5’ppp-dsRNA can be easily monitored using B16 Blue™ ISG cells, an IFN regulatory factor (IRF)-inducible secreted embryonic alkaline phosphatase (SEAP) reporter cell line. B16-Blue™ ISG cells respond to stimulation with 5’ppp-dsRNA and Poly(I:C). The 5’ppp-dsRNA Control (non-phosphorylated) does not induce any response in B16-Blue™ ISG cells. Levels of SEAP is determined by colorimetric measurement using QUANTI-Blue™ and reading optical density (O.D.) at 630-655 nm.

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®

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

Quality control:

• Reporter activity is validated by stimulating the cells with murine IFN-α (mIFN-α), mIFN-β, mIFN-γ, and IRF3 activators, such as poly(dA:dT)/LyoVec™, c-di-GMP and cGAMP.
• The cells are guaranteed mycoplasma-free.

This product is covered by a Limited Use License (See Terms and Conditions).

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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