IL-1β Reporter HEK 293 Cells
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HEK 293 reporter cells for human and murine IL-1β cytokines
3-7 x 10e6 cells
IL-1Beta Reporter Cells
HEK-Blue™ IL-1β cells were engineered from the human embryonic kidney HEK 293 cell line to detect bioactive interleukin-1β (IL-1β) 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. They can also be used for screening anti-IL-1β antibodies.
IL-1β is a soluble pro-inflammatory cytokine that plays a critical role in the host response to infection and injury . It is synthesized as a pro-IL-1β zymogen by activated macrophages and must be cleaved by caspase-1 to generate its mature form . 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 . Due to its role in mediating acute and chronic inflammation, IL-1β has emerged as a therapeutic target for auto-inflammatory diseases [1,4].
Cell line description:
HEK-Blue™ IL-1β cells derive from the HEK-Blue™ Null-1v cell line which expresses an NF-κB/AP-1 inducible secreted embryonic alkaline phosphatase (SEAP) reporter. Binding of IL-1 to its receptor on the surface of HEK-Blue™ IL-1β cells triggers a signaling cascade leading to the activation NF-κB/AP-1 and the subsequent production of SEAP. Levels of SEAP can be easily monitored using QUANTI-Blue™ Solution.
HEK-Blue™ IL-1β cells respond to both human IL-1α and IL-1β, as these cytokines bind to the same receptor, IL-1R1. They also respond to a lesser extent to murine IL-1α and IL-1β. 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.
The parental HEK-Blue™ Null-1v cell line expresses endogenous levels of TLR3, TLR5, and TFNAR, which all signal through NF-κB and AP-1 pathways. The genes encoding these three receptors have been knocked out in HEK-Blue™ IL-1β cells, to avoid interferences between activation of the NF-κB/AP-1 pathways through IL-1β or ligands of TLR3, TLR5 or TNFR. Thus, this reporter cell line can be used to monitor IL-1β production upon TLR3, TLR5, or TFNAR activation with Poly(I:C), Flagellin, or TNFα, respectively.
Features of HEK-Blue™ IL-1β cells:
- Fully functional IL-1β signaling pathway
- Do not respond to human or murine TNF-α
- Do not respond to TLR3 and TLR5 agonists
- Readily assessable SEAP reporter activity
- Functionally tested and guaranteed mycoplasma-free
Applications of HEK-Blue™ IL-1β cells:
- Detection of human IL-1β (0.1 - 100 ng/ml), and to a lesser extent murine IL-1β (10 -1000 ng/ml)
- Detection of IL-1β in inflammasome studies
- Screening of anti-IL-1β antibodies
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.
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 human (h) IL-1β (300 pg/ml), murine (m) IL-1β (300 ng/ml), hIL-1α (3 ng/ml),hIL-1α (3 ng/ml), mIL-1α (30 ng/ml), hTNF-α (100 ng/ml), mTNF-α (100 ng/ml), Flagellin-ST UP (100 ng/ml), or Poly(I:C) HMW (300 ng/ml). 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™ 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.
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™
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
- 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)
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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.Back to the top