Monitoring inflammasome activation by analysing ASC specks and oligomerisation

The inflammasome is a multiprotein cytosolic signalling platform that initiates inflammatory responses upon detection of microbial and danger-associated stimuli. Inflammasome assembly occurs due to the involvement of specific cytosolic pattern recognition receptors (PRRs) in response to pathogens and danger signals in host cells. This causes activation of inflammatory caspases that results in cytokine release and pyroptosis. A central player in inflammasome formation is the adaptor protein ASC (Apoptosis-associated speck-like protein containing a CARD), which bridges activated PRRs to pro-Caspase-1, leading to the formation of the inflammasome complex. This chapter provides detailed protocols for analysing inflammasome assembly by monitoring ASC speck formation using confocal microscopy and biochemically by detecting ASC oligomerisation using DSS-mediated crosslinking. Together, these methods offer complementary insights into spatial organisation and activity of inflammasomes in response to pathogens or cellular damage.