IFN-γ Reporter HEK 293 Cells

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Human Type II IFN Reporter Cells

Signaling pathway in HEK-Blue™ IFN-γ cells

HEK-Blue™ IFN-γ cells were engineered from the human embryonic kidney HEK293 cell line to detect the bioactive human 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

HEK-Blue™ IFN-γ cells were generated by stable transfection with the genes encoding the human STAT1 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. HEK-Blue™ IFN-γ cells were also stably transfected with a STAT1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. 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.

HEK-Blue™ IFN-γ cells respond to human but not murine IFN-γ. Of note, these cells do not respond to either type I IFNs (IFN-α/β) or type III IFNs (IFN-λ) (see figures).

Key Features

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

Applications

• Detection of human IFN-γ
• Quantification of IFN-γ activity in biological samples, such as plasma or serum [2]
• Screening of anti-IFN-γ and anti-IFNGR antibodies
• Screening of small molecule inhibitors of the IFN-γ pathway

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®

Specificity: Detects human IFN-γ 

Detection range:

• Detection range for human IFN-γ: 0.1 ng/ml - 10 ng/ml

Quality Control:

• SEAP reporter activity in response to IFN-γ is validated using functional assays.
• The stability for 20 passages following thawing is confirmed.
• These cells are tested for mycoplasma contamination. 

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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