AMNIS FlowSight Imaging Analysis

PBMCs (1 × 10⁶), stimulated as described above, were fixed with 100 μL of Paraformaldehyde (PFA) (1%) (BDH, United Kingdom), permeabilized with 100 μL of saponin (0.1%) (Life Science VWR, Lutterworth, Leicestershire, LE), and stained with PE-antihuman ASC (clone HASC-71, isotype mouse IgG1, Biolegend, San Diego, CA, United States) for 1 h at room temperature; cells were then washed with PBS, centrifuged at 1,500 rpm for 10 min, resuspended in 50 μL of PBS, and examined using the AMNIS FlowSight Imaging Flow Cytometer (Luminex Corporation Austin, TX). Results were analyzed using an analysis software (IDEAS). The IDEAS image analysis software allows quantification of cellular morphology and fluorescence at different cellular localizations by defining specific cellular regions (masks) and mathematical expressions that uses image pixel data or masks (features).