A flow cytometric assay for ASC speck formation in inflammasome responses (#338)
Inflammasomes are protein complexes that form in
response to infection, cell damage and environmental stress. Inflammasome
activation leads to the processing of proinflammatory cytokines and pyroptotic
cell death through the recruitment and activation of caspase 1, and apoptosis
via caspase 8 activation. A common component of many inflammasomes is the
adaptor molecule “apoptosis associated speck-like protein containing a caspase
activation recruitment domain” (ASC). ASC is normally spread throughout the cell,
however, upon inflammasome activation the vast majority of ASC is recruited
into a single dense speck in the cytosol. We considered that the dramatic
relocalisation of ASC should be detectable by flow cytometry. An assay has been
developed that differentiates cells with ASC specks from cells with diffuse ASC
by comparing time of flight parameters (height, width and integral/area) of the
fluorescence pulse generated as a cell passes through the laser beam. This
analysis has been applied to native ASC within immunostained macrophages and
monocytes, as well as to ASC-GFP overexpressed in HEK293 cells. Applications for this method of speck
detection include examination of early events in inflammasome formation, and
detection of inflammasome responses within mixed cell populations using lineage
markers. In addition this will allow dissection of structural requirements for
ASC speck formation using cells reconstituted for inflammasome formation.