IFN-γ Reporter HEK 293 Cells

HEK-Blue™ IFN-γ Cells signaling pathway

Human Type II IFN Reporter Cells

HEK-Blue™ IFN-γ cells were engineered from the human embryonic kidney HEK 293 cell line to detect the bioactive human type II interferon IFN-γ by monitoring the activation of the JAK/STAT1 pathway.
IFN-γ is a pleiotropic cytokine with anti-viral, anti-tumor, and immunomodulatory functions. It binds a heterodimeric receptor consisting of two subunits, IFNGR1 and IFNGR2, associated with JAK1 and JAK2 [1].

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Description

HEK-Blue™ IFN-γ cells were generated by stable expression of the genes encoding the human STAT1 together with a STAT1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter to obtain a fully active type II IFN signaling pathway. The other genes of the pathway (IFNGR1, IFNGR2, JAK1, and JAK2) are naturally expressed by these cells. STAT1-dependent SEAP activity is readily assessable in the supernatant using QUANTI-Blue™ Solution, a detection reagent. This activation of HEK-Blue™ IFN-γ cells can be blocked with a neutralizing monoclonal antibody, such as anti-hIFN-γ-IgA. Of note, HEK-Blue™ IFN-γ cells do not respond to human type I and type III IFNs (IFN-α/β/λ; see figures).

Key Features

• Fully functional IFN-γ signaling pathway
• Strong response to human IFN-γ
• Unresponsive to murine (m) IFN-γ
• Unresponsive to  IFN-α/β (type I IFN) and IFN-λ (type III IFN)
• Readily assessable SEAP reporter activity

Applications

• Detection of human IFN-γ
• Screening of anti-IFN-γ and anti-IFNGR antibodies
• Quantification of IFN-γ activation in biological samples, such as plasma or serum [2]

References:

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. Gómez-Bañuelos E, et al., 2024. Uncoupling interferons and the interferon signature explain clinical and transcriptional subsets in SLE. medRxiv. 2023.08.28.23294734

Figures

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

Response of HEK-Blue™ IFN-γ cells to a panel of cytokines. Cells were stimulated with various human recombinant cytokines: 100 U/ml hIFN-α2b or hIFN-β-1a, 1 ng/ml hIFN-γ, 10 ng/ml mIFN-γ, 100 ng/ml IL-28a, or 10 ng/ml hTNF-α. After overnight incubation, SEAP activity was assessed using QUANTI-Blue™ Solution. The OD at 630 nm is shown as mean ± SEM.

Dose-dependent inhibition of HEK-Blue™ IFN-γ cell response using Anti-IFN-γ-IgA. A serial dilution of Anti-IFN-γ-IgA monoclonal antibody (mAb) was incubated with 0.3 ng/ml of recombinant human IFN-γ for 30 minutes prior to the addition of the HEK-Blue™ IFN-γ cells. After overnight incubation, SEAP activity in the cell culture supernatant was determined using QUANTI-Blue™ Solution, a SEAP detection reagent. Data are presented as percentage of neutralization (mean).

Specifications

Antibiotic resistance: Blasticidin, Zeocin®

Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v)  heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml Normocin™

Guaranteed mycoplasma-free

Detection range:

• 0.1 - 10 ng/ml for human IFN-γ 

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 Blasticidin (10 mg/ml)
• 1 ml of Zeocin® (100 mg/ml)
• 1 ml 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), a Type II interferon, is secreted from CD4+ T-helper 1 (Th1) cells and activated natural killer (NK) cells. It plays a role in activating lymphocytes to enhance anti-microbial and anti-tumor effects [1-3]. In addition, IFN-γ 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 forms homodimers and are translocated to the nucleus where they bind interferon-gamma-activated sites (GAS) in the promoter of IFN-γ 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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