Detection of ASC Speck Formation by Flow Cytometry and Chemical Cross-linking
Assembly of a relatively large protein aggregate or “speck” formed by the adaptor protein ASC is a common downstream step in the activation of most inflammasomes. This unique feature of ASC allows its visualization by several imaging techniques and constitutes a reliable and feasible readout for inflammasome activation in cells and tissues. We have previously described step-by-step protocols to generate immortalized cell lines stably expressing ASC fused to a fluorescent protein for measuring inflammasome activation by confocal microscopy, and immunofluorescence of endogenous ASC in primary cells. Here, we present two more methods to detect ASC speck formation: (1) Assessment of ASC speck formation by flow cytometry; and (2) Chemical cross-linking of ASC followed by immunoblotting. These methods allow for the discrimination of inflammasome-activated versus non-activated cells, the identification of lineage-specific inflammasome activation in complex cell mixtures, and sorting of inflammasome-activated cells for further analysis.
KeywordsInnate immunity Inflammasome Inflammation Cell signaling Macrophages Flow cytometry Pulse-shape analysis
We would like to thank Dr. Elmar Endl, and Peter Wurst from the flow cytometry core facility of the University Clinics Bonn for valuable discussion during the development of the technique and their continuous support. BF is supported by grants from the Brigitte und Dr. Konstanze Wegener-Stiftung, Germany and Internal Seed Funding Program of the Medical faculty of the University of Bonn (BONFOR).
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