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Tweedell, RE;Malireddi, RKS;Kanneganti, TD;
Inflammasomes are multimeric heterogeneous mega-Dalton protein complexes that play key roles in the host innate immune response to infection and sterile insults. Assembly of the inflammasome complex following infection or injury begins with the oligomerization of the upstream inflammasome-forming sensor and proceeds through a multistep process of well-coordinated events and downstream effector functions. Together, these steps enable elegant experimental readouts with which to reliably assess the successful activation of the inflammasome complex and cell death. Here, we describe a comprehensive protocol that details several in vitro (in bone marrow-derived macrophages) and in vivo (in mice) strategies for activating the inflammasome and explain how to subsequently assess multiple downstream effects in parallel to unequivocally establish the activation status of the inflammasome and cell death pathways. Our workflow assesses inflammasome activation via the formation of the apoptosis-associated speck-like protein containing a CARD (ASC) speck; cleavage of caspase-1 and gasdermin D; release of IL-1?, IL-18, caspase-1, and lactate dehydrogenase from the cell; and real-time analysis of cell death by imaging. Analyses take up to ~24 h to complete. Overall, our multifaceted approach provides a comprehensive and consistent protocol for assessing inflammasome activation and cell death.