IFN-α/β Reporter HEK 293 Cells

Human Type I IFN Reporter Cells

HEK-Blue™ IFN-α/β Cells signaling

HEK-Blue™ IFN-α/β cells allow the detection of bioactive human type I interferons (i.e. IFN-α and IFN-β) by monitoring the activation of the ISGF3 pathway. IFN-α and IFN-β are important anti-viral cytokines that also have anti-proliferative and immunomodulatory functions [1, 2]. These cytokines bind a cell-surface receptor, composed of two subunits, IFNAR1 and IFNAR2, which are associated with TyK2 and JAK1, respectively [1]. Upon binding to this receptor, type I IFNs trigger the JAK/STAT/ISGF3 pathway.

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Cell line description:

HEK-Blue™ IFN-α/β cells were generated by stable transfection of the human embryonic kidney (HEK)-293 cells with the human STAT2 and IRF9 genes to obtain a fully active type I IFN signaling pathway. The other genes of the pathway (IFNAR1, IFNAR2, JAK1, TyK2, and STAT1) are naturally expressed by these cells. The cells feature an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-α/β inducible ISG54 promoter. 

Stimulation of HEK-Blue™ IFN-α/β cells with human IFN-α or IFN-β activates the JAK/STAT/ISGF3 pathway and subsequently induces the production of SEAP. Levels of SEAP are readily assessable in the supernatant using QUANTI-Blue™ Solution. Additionally, the activation of HEK-Blue™ IFN-α/β cells with human IFN-α can be blocked with a neutralizing monoclonal antibody, such as anti-hIFN-α-IgG. Of note, these cells respond to a low extent to type III IFNs (IFN-λ) and poorly to type II IFN (IFN-γ).

Features of HEK-Blue™ IFN-α/β cells:

• Fully functional IFN-α/β signaling pathway
• Poor response to human IFN-γ (type II IFN) and IFN-λ (type III IFN)
• Readily assessable SEAP reporter activity
• Functionally tested and guaranteed mycoplasma-free

Applications of HEK-Blue™ IFN-α/β cells:

• Detection of human IFN-α and IFN-β 
• Screening of anti-hIFN-α or anti-hIFN-β antibodies

References:

1. Schreiber G. 2017. The molecular basis for differential type I interferon signaling. J. Biol. Chem. 292:7285-94.
2. McNab F. et al., 2015. Type I interferons in infectious disease. Nat Rev Immunol. 15(2):87-103.

Figures

Dose-response of HEK-Blue™ IFN-α/β cells to recombinant type I IFNs. Cells were stimulated with increasing concentrations of recombinant human IFN-α (hIFN-α) and IFN-β. After overnight incubation, the ISGF3 response 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-α/β cells to a panel of cytokines. Cells were stimulated with various human recombinant cytokines: 1000 U/ml IFN-α or IFN-β, 300 ng/ml IFN-γ, 10 ng/ml IL-28 or TGF-β, and 100 ng/ml IL-1β, IL-4, IL-6, IL-13, IL-18 or TNF-α. After overnight incubation, SEAP activity was assessed using QUANTI‑Blue™ Solution. The optical density (OD) at 630 nm is shown as mean ± SEM.

Dose-dependent inhibition of HEK-Blue™ IFN-α/β cell response using Anti-IFN-α IgG. A serial dilution of Anti-IFN-α was incubated with 500 U/ml of recombinant human IFN-α2 for 30 minutes prior to the addition of the HEK-Blue™ IFN-α/β cells. After overnight incubation, the ISGF3 response was determined using QUANTI-Blue™ Solution, a SEAP detection reagent. Data are presented as a 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

Detects human type I interferons:

• Detection range for human IFN-α: 1 - 10³ IU/ml
• Detection range for human IFN-β: 3 - 10³ IU/ml

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

Type I interferons, in particular interferon-alpha (IFN-α) and interferon beta (IFN-β), play a vital role in host resistance to viral infections [1, 2]. The type I IFN family is a multi-gene cytokine family that encodes 13 partially homologous IFN-α subtypes in humans (14 in mice), a single IFN-β, and several poorly defined single gene products (IFN-ɛ, IFN-τ, IFN-κ, IFN-ω, IFN-δ, and IFN-ζ) [1, 2].  IFN-α and IFN-β are the best-defined and most broadly expressed type I IFNs [2].

IFN-β and all of the IFN-α subtypes bind to a heterodimeric transmembrane receptor composed of the subunits IFNAR1 and IFNAR2 which are associated with the tyrosine kinases Tyk2 and Jak1 (Janus kinase 1) respectively. These kinases phosphorylate STAT1 and STAT2 which then dimerize and interact with IFN regulatory factor 9 (IRF9), leading to the formation of the ISGF3 complex. ISGF3 binds to IFN-stimulated response elements (ISRE) in the promoters of IFN-stimulated genes (ISG) to regulate their expression. 

Stimulation of HEK-Blue™ IFN-α/β cells with human IFN-α or IFN-β activates the JAK/STAT/ISGF3 pathway and subsequently induces the production of SEAP.

1. Schreiber G. 2017. The molecular basis for differential type I interferon signaling. J. Biol. Chem. 292:7285-94.
2. McNab F. et al., 2015. Type I interferons in infectious disease. Nat Rev Immunol. 15(2):87-103.

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