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Updated: Mar 2, 2026
Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Published on: May 22, 2014
Reconstruction of the Mouse Inflammasome System in HEK293T Cells
Hexin Shi1, Anne Murray1, Bruce Beutler1
1Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
Abstract:
The NLRP3 (NLR family, Pyrin domain containing 3) inflammasome is a multiprotein complex comprised of NLRP3, pro-caspase-1, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and the protein kinase NIMA related kinase 7 (NEK7) (Shi et al., 2016; He et al., 2016; Schmid-Burgk et al., 2016). When cells are exposed to microbes and/or danger signals, the inflammasome assembles and serves as a platform for the activation of caspase-1. Caspase-1 activation promotes the processing and secretion of the pro-inflammatory cytokines interleukin-1β (IL-1β), IL-18, and IL-33 as well as pyroptosis induction (Gross et al., 2011; Arend et al., 2008), which elicit inflammatory responses. Here, we describe how to co-transfect the NLRP3 inflammasome components into HEK293T cells, which enables inflammasome activation and the production of IL-1β upon stimulation with nigericin.
Insights
This study details co-transfecting NLRP3 inflammasome components into HEK293T cells. This method successfully activates the inflammasome and induces interleukin-1β production upon nigericin stimulation, aiding inflammatory response research.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- The NLRP3 inflammasome is a multiprotein complex crucial for innate immunity.
- It activates caspase-1, leading to pro-inflammatory cytokine secretion and pyroptosis.
- NLRP3 inflammasome activation is triggered by microbial and danger signals.
Purpose of the Study:
- To establish a method for co-transfecting NLRP3 inflammasome components into HEK293T cells.
- To demonstrate inflammasome activation and interleukin-1β production in this system.
- To provide a model for studying NLRP3 inflammasome-mediated inflammatory responses.
Main Methods:
- Co-transfection of NLRP3, pro-caspase-1, ASC, and NEK7 into HEK293T cells.
- Stimulation of transfected cells with nigericin.
- Assessment of inflammasome activation and IL-1β production.
Main Results:
- Successful co-transfection and assembly of the NLRP3 inflammasome components.
- Nigericin stimulation induced caspase-1 activation and IL-1β secretion.
- The HEK293T cell system effectively models NLRP3 inflammasome activation.
Conclusions:
- Co-transfection of NLRP3 inflammasome components into HEK293T cells is a viable method for studying inflammasome activation.
- This system facilitates the investigation of IL-1β production and inflammatory pathways.
- The established protocol aids research into the role of NLRP3 inflammasome in various biological processes.
