A flow cytometric assay for ASC speck formation in inflammasome responses — ASN Events

A flow cytometric assay for ASC speck formation in inflammasome responses (#338)

Sara J Thygesen 1 , David Sester 1 , Vita Sagulenko 1 , Jasmyn Cridland 1 , Geoff Osborne 2 , Kate Schroder 3 , Kate Stacey 1
  1. School of Chemistry and Molecular Biosciences , The University of Queensland, Brisbane, Queensland, Australia
  2. Queensland Brain Institute, The University of Queensland , Brisbane, Queensland , Australia
  3. Institute for Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia
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.