Haitao Guo1, Jenny P-Y Ting1,2,3. 1. Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. 2. Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. 3. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Abstract
This article presents assays that allow induction and measurement of activation of different inflammasomes in mouse macrophages, human peripheral blood mononuclear cell (PBMC) cultures, and mouse peritonitis and endotoxic shock models. Basic Protocol 1 describes how to prime the inflammasome in mouse macrophages with different Toll-like receptor agonists and TNF-α; how to induce NLRP1, NLRP3, NLRC4, and AIM2 inflammasome activation by their corresponding stimuli; and how to measure inflammasome activation-mediated maturation of interleukin (IL)-1β and IL-18 and pyroptosis. Since the well-established agonists for NLRP1 are inconsistent between mice and humans, Basic Protocol 2 describes how to activate the NLRP1 inflammasome in human PBMCs. Basic Protocol 3 describes how to purify, crosslink, and detect the apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome. Formation of the ASC pyroptosome is a signature of inflammasome activation. A limitation of ASC pyroptosome detection is the requirement of a relatively large cell number. Alternate Protocol 1 is provided to stain ASC pyroptosomes using an anti-ASC antibody and to measure ASC specks by fluorescence microscopy in a single cell. Intraperitoneal injection of lipopolysaccharides (LPS) and inflammasome agonists will induce peritonitis, which is seen as an elevation of IL-1β and other proinflammatory cytokines and an infiltration of neutrophils and inflammatory monocytes. Basic Protocol 4 describes how to induce NLRP3 inflammasome activation and peritonitis by priming mice with LPS and subsequently challenging them with monosodium urate (MSU). The method for measuring cytokines in serum and through peritoneal lavage is also described. Finally, Alternate Protocol 2 describes how to induce noncanonical NLRP3 inflammasome activation by high-dose LPS challenge in a sepsis model.
This article presents assays that allow induction and measurement of activation of different inflammasomes in mouse macrophages, human peripheral blood mononuclear cell (PBMC) cultures, and mouseperitonitis and endotoxic shock models. Basic Protocol 1 describes how to prime the inflammasome in mouse macrophages with different Toll-like receptor agonists and TNF-α; how to induce NLRP1, NLRP3, NLRC4, and AIM2 inflammasome activation by their corresponding stimuli; and how to measure inflammasome activation-mediated maturation of interleukin (IL)-1β and IL-18 and pyroptosis. Since the well-established agonists for NLRP1 are inconsistent between mice and humans, Basic Protocol 2 describes how to activate the NLRP1 inflammasome in human PBMCs. Basic Protocol 3 describes how to purify, crosslink, and detect the apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome. Formation of the ASC pyroptosome is a signature of inflammasome activation. A limitation of ASC pyroptosome detection is the requirement of a relatively large cell number. Alternate Protocol 1 is provided to stain ASC pyroptosomes using an anti-ASC antibody and to measure ASC specks by fluorescence microscopy in a single cell. Intraperitoneal injection of lipopolysaccharides (LPS) and inflammasome agonists will induce peritonitis, which is seen as an elevation of IL-1β and other proinflammatory cytokines and an infiltration of neutrophils and inflammatory monocytes. Basic Protocol 4 describes how to induce NLRP3 inflammasome activation and peritonitis by priming mice with LPS and subsequently challenging them with monosodium urate (MSU). The method for measuring cytokines in serum and through peritoneal lavage is also described. Finally, Alternate Protocol 2 describes how to induce noncanonical NLRP3 inflammasome activation by high-dose LPS challenge in a sepsis model.