IL-18 Reporter HEK 293 Cells

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IL-18 Reporter Cells

Signaling pathway in HEK-Blue™ IL-18 cells

HEK-Blue™ IL-18 cells were engineered from the human embryonic kidney 293 (HEK293) cell line to detect bioactive interleukin-18 (IL-18) by monitoring the activation of the NF-κB and AP-1 pathways. These cells are useful to monitor IL-18 secretion in inflammasome activation studies. In addition, these cells can be used for screening antibodies or small molecule inhibitors targeting the IL-18 pathway.

IL-18 is a pro-inflammatory cytokine that causes a wide variety of biological effects associated with infection, inflammation, and autoimmune processes [1, 2]. 

More details

Cell line description

HEK-Blue™ IL-18 cells were generated by stable transfection with the genes encoding the IL-18 receptor (IL-18R) and IL-18 receptor accessory protein (IL-18RAP). These cells were further transfected with an NF-κB/AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. The binding of IL-18 to its heterodimeric IL-18 receptor triggers a signaling cascade leading to NF-κB/AP-1 activation and the subsequent production of SEAP. This can be readily assessed in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent.

HEK-Blue™  IL-18 cells are highly sensitive to human (h) IL-18 and also respond to a lesser extent to murine (m) IL-18 (see figures). These cells are non‑responsive to hIL-1β, hIL-33, hIFN-α, hIFN-β , hIFN-γ, and hTNF-α (see figures).

Key Features

• Fully functional IL-18 signaling pathway
• Readily assessable NF-κB/AP-1-inducible SEAP reporter activity
• Strong response to human (h) IL-18 and lower response to murine (m) IL-18
• No response to hTNF-α and hIL-1β

Applications

• Detection and quantification of hIL-18 and mIL-18 activity
• Screening of anti-IL-18 and anti-IL-18 receptor antibodies
• Screening of small molecule inhibitors of the IL-18 pathway

References:

1. Yasuda K. et al., 2019. Interleukin-18 in health and disease. Int J Mol Sci. 20(3).
2. Dinarello CA. et al., 1998. Overview of interleukin-18: more than an interferon-gamma inducing factor. J Leukoc Biol. 63(6):658-64.
3. Gracie JA. et al., 2003. Interleukin-18. J Leukoc Biol.3(2):213-24.
4. Kojima H. et al., 1998. Interleukin-18 activates the IRAK-TRAF6 pathway in mouse EL-4 cells. BBRC 244(1):183-6.

Figures

Cells were stimulated with increasing concentrations of recombinant human IL-18 (hIL-18) and murine  IL-18 (mIL-18). After overnight incubation, the NF-κB/AP-1 response was determined using QUANTI-Blue™ Solution, a SEAP detection reagent, and reading the optical density (OD) at 630 nm.

Response of HEK‑Blue™ IL‑18 cells to a panel of cytokines. Cells were stimulated with various human and murine recombinant cytokines: 1  ng/ml of hIL-18, mIL-18, 10  ng/ml of hIL-1β, hIL-33, hIFN-γ, hTNF-α, or 102 U/ml hIFN-α or hIFN-β. After overnight incubation, SEAP activity was assessed using QUANTI-Blue™ Solution. The optical density (OD) at 630 nm is shown as mean ± SD.

Dose-dependent inhibition of HEK-Blue™ IL-18 cellular response using a neutralizing antibody against hIL-18. The antibody was incubated with hIL-18 (10 pg/ml) for 2 hours prior to the addition of HEK-Blue™ IL‑18 cells. After overnight incubation, SEAP activity in the cell culture supernatant was assessed using QUANTI‑Blue™ Solution. Data represent % of maximal reporter activity without the anti-hIL18 antibody.

Specifications

Antibiotic resistance: Blasticidin, Hygromycin B, 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 and mouse IL-18

Detection range:

• hIL-18: 10 pg - 1 ng/ml
• mIL-18: 3 ng - 100 ng/ml 

Quality Control:

• SEAP reporter activity in response to IL-18 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
• 2 x 1 ml of HEK-Blue™ Selection (250x concentrate)
• 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

Interleukin-18 (IL-18), formerly called interferon-γ (IFN-γ) inducing factor, is a pro-inflammatory cytokine that causes a wide variety of biological effects associated with infection, inflammation, and autoimmune processes [1, 2]. More specifically, IL-18 induces IFN-γ production and contributes to T-helper 1 (Th1) cell polarization.

IL-18 is produced by macrophages and other cells, as a pro-protein which is proteolytically processed to its active form by caspase 1, an enzyme that is activated within the inflammasome multiprotein complex. It binds to a heterodimeric receptor consisting of IL-18R and IL-18 receptor accessory protein (IL-18RAP). Upon binding, IL-18 activates NF-κB and AP-1 via signaling pathways that involve TRAF-6 [3, 4].

1. Yasuda K. et al., 2019. Interleukin-18 in health and disease. Int J Mol Sci. 20(3).
2. Dinarello CA. et al., 1998. Overview of interleukin-18: more than an interferon-gamma inducing factor. J Leukoc Biol. 63(6):658-64.
3. Gracie JA. et al., 2003. Interleukin-18. J Leukoc Biol.3(2):213-24.
4. Kojima H. et al., 1998. Interleukin-18 activates the IRAK-TRAF6 pathway in mouse EL-4 cells. BBRC 244(1):183-6.

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