Imaging of Inflammasome Speck Formation in Living Cells.

The detection of pathogen- or danger-associated molecular patterns during an inflammatory injury triggers the activation of cytosolic sensors known as inflammasomes. Once stimulated, these protein complexes can connect to the adaptor protein ASC, which in turn recruits the effector enzyme caspase-1, forming a polymeric structure known as ASC speck. This protein scaffold is responsible for processing cytokines of the IL-1 family into their active forms and evoking the cleavage of gasdermin D, ultimately leading to cell death by pyroptosis. Due to its micrometric size, the specks are used as a readout for inflammasome activation and for the better comprehension of this important immune pathway. In this chapter, a detailed protocol is presented for the study of the formation of inflammasome specks in living cells using confocal microscopy.