Hallmarks of NLRP3 inflammasome activation include the cleavage and secretion of the mature forms of caspase-1, IL-1β, and IL-18 and aggregation of ASC into "specks." We have previously shown that macrophage migratory inhibitory factor (MIF) directly regulates activation of the NLRP3 inflammasome, inhibiting the release of interleukin (IL)-1α, IL-1β, and IL-18. Here we present protocols for studying activation of the NLRP3 inflammasome in human and mouse macrophages and peripheral blood mononuclear cells (PBMCs). These protocols can also be applied to different cell types, such as fibroblasts, neutrophils, endothelial cells, and epithelial cells, although further optimization may be required for each. We also cover the stimulation of macrophages with established NLRP3 inflammasome activators.
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