It is composed of the receptor or sensor molecule NOD-like receptors (NLR) or Absent in melanoma (AIM2)-like receptors, adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and pro-caspase-1, the inflammasome platform regulates caspase-1 activation and subsequent interleukin-1β (IL-1β) and IL-18 maturation and inflammatory cell death, pyroptosis ( Lamkanfi & Dixit, 2014). Inflammasomes are supramolecular complexes that play the pivotal role in mounting inflammation. Finally, an alternative protocol describes how to induce noncanonical NLRP3 inflammasome activation by high-dose LPS challenging in a sepsis model. Basic protocol 4 describes how to induce NLRP3 inflammasome activation and peritonitis by priming mice with LPS and subsequent challenging with MSU The method for measuring cytokines in serum and peritoneal lavage is also described. Intraperitoneal injection of LPS plus inflammasome agonists will induce peritonitis that is featured by an elevation of IL-1β in addition to other proinflammatory cytokines and an infiltration of neutrophils and inflammatory monocytes. An alternative protocol is provided to stain ASC pyroptosome by the anti-ASC antibody and measure ASC speck by fluorescence microscopy in the single cell. A limitation of ASC pyroptosome detection is requiring a relative large cell number. Formation of ASC pyroptosome is a signature of inflammasome activation. Basic protocol 3 describes how to purify, crosslink, and detect ASC pyroptosome. Since the well-established agonists for NLRP1 are inconsistent between mice and human, basic protocol 2 describes how to activate NLRP1 inflammasome in human PBMCs. Basic protocol 1 describes how to prime the inflammasome in mouse macrophages with different Toll-like receptor agonists and TNF-α, followed by the induction of NLRP1, NLRP3, NLRC4, and AIM2 inflammasomes activation by their corresponding stimuli, and subsequent measurement of inflammasome activation-mediated maturation of IL-1β and IL-18 and pyroptosis. This unit presents assays that allow induction and measurement of different inflammasomes activation in the mouse macrophages and human PBMC (peripheral blood mononuclear cell) cultures and mouse peritonitis and endotoxic shock models.
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December 2022
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