Cellular immunofluorescence staining was performed as previously reported (Hwang et al., 2019 (link)). Cells seeded on slides were fixed with 4% PFA for 20 min at room temperature, washed with PBS, and blocked in 10% bovine serum albumin (Cat#4240GR100, BioFroxx) for 2 h in before being probed with p65 (1:500, Cat#8242, Cell Signaling Technology) or ASC (1:500, Cat#67824s, Cell Signaling Technology) antibodies. Thereafter, cells were incubated with donkey-anti-rabbit 488 (1:500, 711-545-152, Jackson) at room temperature and protected from light for 1 h. Images were acquired by Leica TSC SP8 confocal microscope. For p65 nuclear translocation analysis, Image J was used to frame nuclei and then the nuclear p65 signal fluorescence intensity was calculated. For ASC speck analysis, 5–6 slides were analyzed in each group, and ∼6–10 fields were analyzed for each slide. The percentage of ASC speck-positive cells as well as total cells per field was calculated using the Fiji software (Nagar et al., 2021 (link)).
For pyroptosis-like morphology analysis, images were acquired by Leica DMIL microscope (Leica) with the phase-contrast mode. Images were statistically analyzed using the Fiji software. Three wells (20 fields per well) of each group were detected. Percentage of pyroptosis-like blebs in total cells per field was calculated (Herr et al., 2020 ).
For pyroptosis-like morphology analysis, images were acquired by Leica DMIL microscope (Leica) with the phase-contrast mode. Images were statistically analyzed using the Fiji software. Three wells (20 fields per well) of each group were detected. Percentage of pyroptosis-like blebs in total cells per field was calculated (Herr et al., 2020 ).
