B16-Blue™ IFN-γ Cells

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Murine Type II IFN Reporter Cells

Signaling pathway in B16-Blue™ IFN-γ cells

B16-Blue™ IFN-γ cells were engineered from the murine B16 melanoma cell line to detect bioactive murine type II interferon IFN-γ by monitoring the activation of the JAK/STAT1 pathway. In addition, these cells can be used for screening antibodies or small molecule inhibitors targeting the IFN-γ pathway.

IFN-γ is a pleiotropic cytokine with anti-viral, anti-tumor, and immunomodulatory functions [1].

 More details

Cell line description

B16-Blue™ IFN-γ cells were generated by stable transfection with the secreted embryonic alkaline phosphatase (SEAP) reporter under the control of the ISG54 promoter. This promoter comprises IFN-stimulated response elements (ISRE) that are recognized by the ISGF3 complex. The binding of IFN-γ to its receptor triggers a signaling cascade leading to the activation of STAT1 and the subsequent production of SEAP. This can be readily assessed in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent.
B16-Blue™ IFN-γ cells respond specifically to murine (m) IFN-γ and do not respond to human (h) IFN-γ. Of note they do not respond to mIFN-α/β (see figures).

Key features

• Fully functional murine IFN-γ signaling pathway
• Readily assessable STAT1-inducible SEAP reporter activity
• Strong response to murine IFN-γ
• No response to human IFN-γ
• No response to murine IFN-α/β (type I IFNs)

Applications

• Detection of murine IFN-γ
• Screening of anti-IFN-γ and anti-IFNGR antibodies
• Screening of small molecule inhibitors of the IFN-γ pathway

Reference:

1. Ivashkiv L.B., 2018. IFNγ: signalling, epigenetics and roles in immunity, metabolism, disease and cancer immunotherapy. Nat Rev Immunol. 18(9):545-558.
2. Platanias LC., 2005. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol. 5(5):375-86.

Figures

Dose-response of B16-Blue™ IFN-γ cells to recombinant murine IFN-γ.
Cells were stimulated with increasing concentrations of recombinant murine IFN-γ. After overnight incubation, the ISGF3 response was determined using QUANTI-Blue™ Solution, a SEAP detection reagent, and reading the optical density (OD) at 630 nm. The OD at 630 nm is shown as mean ± SEM.

Response of  B16-Blue™ IFN-γ cells to a panel of cytokines.
Cells were stimulated with various human and murine recombinant cytokines: 10 ng/ml of mIFN-γ, mIFN-λ, hIFN-γ and 1000U/ml of mIFN-αA (also known as mIFN-α3), mIFN-β, hIFN-α2a, hIFN-β. After overnight incubation, SEAP activity was assessed using QUANTI-Blue™ Solution. The OD at 630 nm is shown as mean ± SEM.

Specifications

Detection range for murine IFN-γ: 0.1 ng - 1 µg/ml for mIFN-γ

Antibiotic resistance: Zeocin®

Quality control:

• B16-Blue™ IFN-γ cells were stimulated with murine IFN-α, IFN-β and IFN-γ. The cells produce SEAP only in response to murine IFN-γ
• 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

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)

Details

Interferon-gamma (IFN-γ) is the sole member of the type II IFN family. It is secreted from CD4+ Th1 cells and activated NK cells. It plays a role in activating lymphocytes to enhance anti-microbial and anti-tumor effects [1-]. In addition, it plays a role in regulating the proliferation, differentiation, and response of lymphocyte subsets.

IFN-γ exerts its action by first binding to a heterodimeric receptor consisting of two chains, IFNGR1 and IFNGR2, causing its dimerization and the activation of specific Janus family kinases (JAK1 and JAK2) [4, 5]. Two STAT1 molecules then associate with this ligand-activated receptor complex and are activated by phosphorylation. Activated STAT1 molecules form homodimers and are translocated to the nucleus where they bind IFN-stimulated response elements (ISRE) in the promoter of IFN inducible genes.

1. Ivashkiv L.B., 2018. IFNγ: signalling, epigenetics and roles in immunity, metabolism, disease and cancer immunotherapy. Nat Rev Immunol. 18(9):545-558.
2. Shtrichman R. & Samuel CE., 2001. The role of gamma interferon in antimicrobial immunity. Curr Opin Microbiol. 4(3):251-9.
3. Sato A. et al., 2006. Antitumor activity of IFN-lambda in murine tumor models. J Immunol. 176(12):7686-94.
4. Platanias L.C., 2005. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol. 5(5):375-86.
5. Schroder K. et al., 2004. Interferon-gamma: an overview of signals, mechanisms, and functions. J Leukoc Biol. 75(2):163-89.

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