Pyroptosis Markers in Human Primary Specimens: Quantification of Intracellular ASC Specks by Imaging Flow Cytometry and Extracellular Oxidized Mitochondrial by ELISA.

Pyroptosis is an immunological response to infection and cellular stresses initiated by inflammasome oligomerization resulting in the release of pro-inflammatory factors including cytokines and other immune stimuli into the extracellular matrix. In order to understand the role of inflammasome activation and subsequent pyroptosis in human infection and disease pathogenesis and to explore markers of these signaling events as potential disease or response biomarkers, we must utilize quantitative, reliable, and reproducible assays to readily investigate these pathways in primary specimens. Here, we describe two methods using imaging flow cytometry for evaluation of inflammasome ASC specks in homogeneous peripheral blood monocytes and in bulk, heterogeneous peripheral blood mononuclear cells. Both methods can be applied to assess speck formation as a biomarker for inflammasome activation in primary specimens. Additionally, we describe the methods for quantification of extracellular oxidized mitochondrial DNA from primary plasma samples, serving as a proxy for pyroptosis. Collectively, these assays may be utilized to determine pyroptotic influences on viral infection and disease development or as diagnostic aids and response biomarkers.