Human IL-1α & IL-1β Reporter HEK 293 Cells

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Human IL-1 Reporter Cells

Signaling pathway in HEK-Blue™ IL-1β cells

HEK-Blue™ IL-1β cells were engineered from the human embryonic kidney HEK293 cell line to detect bioactive human interleukin-1 β (IL-1β) in various biological samples (cell culture supernatant and serum) by monitoring the activation of the NF-κB and AP-1 pathways. These cells are useful to monitor IL-1β secretion in inflammasome activation studies. Additionally, HEK-Blue™ IL-1β detect bioactive human IL-1α. HEK-Blue™ IL-1β cells can also be used for screening antibodies or small molecule inhibitors targeting the IL-1 pathway. 

Both IL‑1α and IL-1β are secreted pro‑inflammatory cytokine that play a critical role in immune responses and inflammation [1].

More details

Cell line description

HEK-Blue™ IL-1β cells endogenously express the human (h) IL-1 receptor, which binds both IL-1α and IL-1β. They were transfected with a NF-κB/AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. The binding of IL-1α or IL-1β to its 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-1β cells respond to hIL-1α and hIL-1β (see figures). They also respond to a lesser extent to murine (m) IL-1α and mIL-1β (see figures). The specificity of the HEK-Blue™ IL-1β cells for the detection of IL-1α or IL-1β can be confirmed using a neutralizing antibody against human IL-1α or IL-1β, such as anti-hIL-1α-IgG and anti-hIL-1β-IgG. Of note, these cells do no respond to hTNF-α or mTNF-α (see figures).

Key features

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

Applications

• Detection and quantification of hIL-1β activity, and to a lesser extent mIL-1β activity
• Detection of hIL-1β in inflammasome studies
• Screening of anti-IL-1α or -IL-1β antibodies and anti-IL-1 receptor antibodies
• Screening of small molecule inhibitors of the IL-1 pathway

Download our Practical guide on Inflammasomes

Reference:

1. Dinarello C., 2018. Overview of the IL-1 family in innate inflammation and acquired immunity. Immunol Rev. 281(1): 8–27. 

Figures

Stimulation of HEK-Blue™ IL-1β cells by recombinant human and murine IL-1β. After a 24 h incubation, SEAP activity was assessed using QUANTI-Blue™ Solution and reading the optical density (O.D.) at 630 nm.

Stimulation of HEK-Blue™ IL-1β cells by recombinant human and murine IL-1α. After a 24 h incubation, SEAP activity was assessed using QUANTI-Blue™ Solution and reading the optical density (O.D.) at 630 nm.

Specific response of HEK‑Blue™ IL ‑1β cells to IL‑1β and IL‑ 1α cytokines.

Cells were stimulated with 1 ng/ml of hIL‑1β, mIL-1β, hIL-1α, mIL-1α, 100 ng/ml of hTNF-α, mTNF-α, ultrapure flagellin from S. typhimurium (FLA-ST UP) or 300 ng/ml of Poly(I:C) HMW. After overnight incubation, SEAP activity was assessed using QUANTI‑Blue™ Solution. OD at 630 nm is shown as mean ± SEM.

Dose‑dependent inhibition of HEK‑Blue™ IL‑1β cellular response using a neutralizing antibody against hIL‑1β. The anti-hIL1β antibody was incubated with the cells for 30 minutes prior to the addition of hIL-1β (1 ng/ml). 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-hIL1β antibody.

Specifications

Antibiotic resistance: 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 and murine IL-1β (α)

• Detection range for human IL-1β: 100 pg - 100 ng/ml
• Detection range for murine IL-1β: 10 ng - 1 µg/ml

This product is covered by a Limited Use License (See Terms and Conditions).

Contents

• 1 vial containing 3-5 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

IL-1β is a soluble pro-inflammatory cytokine that plays a critical role in the host response to infection and injury [1]. It is synthesized as a pro-IL-1β zymogen by activated macrophages and must be cleaved by caspase-1 to generate its mature form [2]. IL-1β binding to the IL-1R1 receptor triggers the formation of the IL-1R1/IL-1R3/MyD88 complex and induces signaling leading to the activation of the transcription factors NK-κB and AP-1 [3]. Due to its role in mediating acute and chronic inflammation, IL-1β has emerged as a therapeutic target for auto-inflammatory diseases [1,4]. 

1. Dinarello C., 2018. Overview of the IL-1 family in innate inflammation and acquired immunity. Immunol Rev. 281(1): 8–27. 
2. Lopez-Castejon G. & Brough D., 2011. Understanding the mechanism of IL-1β secretion. Cytokine Growth Factor Rev. 22(4):189-95.
3. Weber A. et al., 2010. Interleukin-1 (IL-1) pathway. Sci Signal. 3(105):cm1.
4. Dinarello CA., 2011. Interleukin-1 in the pathogenesis and treatment of inflammatory diseases. Blood. 117:3720–3732.

THP-1/HEK-Blue™ IL-1β Assay

1. Production of IL-1β by THP-1 cells. Typically, THP-1 cells are pre-treated with phorbol 12-myristate acetate (PMA) to become more susceptible to inflammasome activators, then are primed with lipopolysaccharide (LPS). These treatments induce the production of pro-IL-1β, the immature form of IL-1β. Subsequent stimulation with inflammasome inducers, such as ATP, leads to NRLP3 and caspase-1 activation resulting in IL-1β maturation and secretion.

2. Detection of IL-1β by HEK-Blue™ IL-1β cells. IL-1β-containing THP-1 supernatants are added to HEK-Blue™ IL-1β cells leading to NF-κB activation and the subsequent production of SEAP. The presence of SEAP in HEK-Blue™ IL-1β supernatants is assessed using QUANTI-Blue™ Solution, a SEAP detection medium.

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