Flow Imaging of the Inflammasome: Evaluating ASC Speck Characteristics and Caspase-1 Activity

Examining inflammasome-associated speck structures is one of the most preferred and easiest ways to evaluate inflammasome activation. Microscopy-based evaluation of specks is preferable, but this approach is time-consuming and limited to small sample sizes. Speck-containing cells can also be quantitated by a flow cytometric method, time of flight inflammasome evaluation (TOFIE). However, TOFIE cannot perform single-cell analysis such as simultaneously visualizing ASC specks and caspase-1 activity, their location, and physical characteristics. Here we describe the application of an imaging flow cytometry-based approach that overcomes these limitations. Inflammasome and Caspase-1 Activity Characterization and Evaluation (ICCE) is a high-throughput, single-cell, rapid image analysis utilizing the Amnis ImageStream X instrument with over 99.5% accuracy. ICCE quantitatively and qualitatively characterizes the frequency, area, and cellular distribution of ASC specks and caspase-1 activity in mouse and human cells.