Calcein-AM/propidium Iodide (PI) staining assay

Calcein-AM/PI double staining (cat. no. C2015M; Beyotime Institute of Biotechnology) was used to quantify the number of living and dead cells as a cell death assay. Briefly, the macrophages and THP-1 cells (5x10⁵ cells/ml) were mixed with 1X assay buffer and then stained with 2 µM calcein-AM and 4.5 µM PI per well for 30 min at 37˚C. The percentage of positive cells was counted and the image were scanned with a fluorescence microscope (magnification, x100). The number of PI positive cells represented pyroptosis, which was calculated using the following equation: PI positive cells (%)=(PI positive cells/Calcein-AM positive cells) x100%.