title: Measuring inflammasome activation in response to bacterial infection.
abstract: Inflammasomes are multi-protein signaling platforms assembled in response to viral and bacterial pathogens as well as endogenous danger signals. Inflammasomes serve as activation platforms for the mammalian cysteine protease caspase-1, a central mediator of innate immunity. The hallmarks of inflammasome activation are the processing of caspase-1, the maturation and release of interleukin-1β (IL-1β) and the induction of pyroptosis, a lytic inflammatory cell death. This protocol describes methods for studying inflammasome activation in response to bacterial pathogens in bone-marrow derived murine macrophages (BMDMs). In particular, we outline the protocols to measure cytokine maturation by ELISA and pyroptosis by the release of Lactate Dehydrogenase (LDH). In addition, we describe methods to visualize endogenous ASC specks or foci in infected cells and to study the release of processed caspase-1, caspase-11 and mature cytokines into the cell supernatant by Western blotting. General considerations are discussed to design and optimize the infection protocol for the study of inflammasome activation by other bacterial pathogens.
authors.affiliation: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA.
authors.forename: Petr
authors.initials: P
authors.lastname: Broz
authors.forename: Denise M
authors.initials: DM
authors.lastname: Monack
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001424:Bacterial Infections / Q000276:immunology / Q000378:metabolism*; D015153:Blotting, Western; D053468:CARD Signaling Adaptor Proteins; D020170:Caspase 1 / Q000378:metabolism; D002454:Cell Differentiation; D016207:Cytokines / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D058847:Inflammasomes / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D051379:Mice; D018613:Microscopy, Confocal
chemical_list: D051017:Apoptosis Regulatory Proteins; D053468:CARD Signaling Adaptor Proteins; D016207:Cytokines; D003598:Cytoskeletal Proteins; D058847:Inflammasomes; C498534:Pycard protein, mouse; D020170:Caspase 1