Extended Data Fig. 1: ASC speck percentage is increased in cell populations with high NLRP3 expression.

a. Schematic diagram of NLRP3 variants and classification. b. Gating strategy for image-based flow cytometry analysis of ASC speck formation in ASC-BFP HEK293T cells. c. Representative images for ASC speck-negative and positive cells following gating as in b. d. Flow cytometry analysis of ASC speck percentage in cell groups with different levels of NLRP3 expression. e. Gating strategy (left) and representative images (right) for ASC speck percentage in cell populations with low, medium, or high level of NLRP3-GFP expression. Images were created using BioRender. f. Schematic diagram of automated ASC50 analysis progress. Cells were separated into a speck-positive and speck-negative population (left) and then analyzed based on the NLRP3-GFP signal (middle). The ASC percentage was calculated for each population with the same level of GFP expression. The EC50 was calculated based on the ASC percentage curve and the EC50 ratio was generated by comparing the mutant to wild-type (WT) NLRP3 (right). Data were pooled from 4 independent repeats (d, mean and s.e.m. in d) or representative of 3 independent repeats (b, c, e).