Abstract
Inflammasome dysregulation is a hallmark of various inflammatory diseases. Evaluating inflammasome-associated structures (ASC specks) and caspase-1 activity by microscopy is time consuming and limited by small sample size. The current flow cytometric method, time of flight inflammasome evaluation (TOFIE), cannot visualize ASC specks or caspase-1 activity, making colocalization studies of inflammasome components and enzymatic activity impossible. We describe a rapid, high-throughput, single-cell, fluorescence-based image analysis method utilizing the Amnis ImageStreamX instrument that quantitatively and qualitatively characterizes the frequency, area, and cellular distribution of ASC specks and caspase-1 activity in mouse and human cells. Unlike TOFIE, this method differentiates between singular perinuclear specks and false positives. With our technique we also show that the presence of NLRP3 reduces the size of ASC specks, which is further reduced by the presence of active caspase-1. The capacity of our approach to simultaneously detect and quantify ASC specks and caspase-1 activity, both at the population and single-cell level, renders it the most powerful tool available for visualizing and quantifying the impact of mutations on inflammasome assembly and activity.