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Detection of ASC Speck Formation by Flow Cytometry and Chemical Cross-linking

2017; Springer Science+Business Media; Linguagem: Inglês

10.1007/978-1-4939-7519-8_10

1940-6029

Florian Hoß, Verena Rolfes, Mariana Rodrigues Davanso, Tárcio Teodoro Braga, Bernardo S. Franklin,

Heme Oxygenase-1 and Carbon Monoxide

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.