Sester and colleagues performed an FCM method to detect ASC redistribution in myeloid cell defining speck formation by changes in fluorescence peak height and width [2156 (link), 2157 (link)]. This protocol permits to define the true activated inflammasome by assessment ASC speck formation, because until then the detection of inflammasome, activation was made by monitor its end products, IL-1β or IL-18, or activated caspase-1 detection. Notably different pathways can secrete IL-1β and the methods to analyze caspase-1 activity are not always specific, in both cases the outcomes could be inflammasome independent. Despite to ASC speck formation microscopy analysis, used in the past, this methodology is faster, more accurate, and sensitive. Recently a better method to analyze simultaneously ASC speck and caspase-1 activity was performed by Amnis ImageStreamX [2158 (link)]. This protocol eliminates false positive events detected by flow-cytometry method, by specific masks to select only single cell, excluding cells with nonspecific-like aggregation of ASC and also defining ASC speck size. Finally the study analyze the presence and distribution of active caspase-1, detected by FLICA spots, and ASC speck simultaneously, evaluating inflammasome activation.
In this section we show the analysis of inflammasome activation by FlowSight, performed utilizing ASC speck formation in LPS+Nig stimulated-THP1derived macrophage.