Propidium iodide

The iBMDM cell lines reconstituted with wildtype or mutant Gsdmd were seeded in 24-well plates with 4 × 10⁵ cells per well. After overnight culture, cells were washed with PBS before adding balanced salt solutions (25 mM Na-HEPES, pH 7.4, 130 mM NaCl, 4 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2) supplemented with 5 mM D-glucose and 0.1% BSA. Cells were then infected with a Salmonella enterica serotype Typhimurium expressing GFP (ATCC 14028GFP). The infection was carried out at 37°C with an MOI of 10:1 in the presence of 1 μg/ml propidium iodide (Thermo Fisher Scientific, Waltham, MA). The propidium iodide fluorescence (excitation/emission: 533/617 nm) in each well was measured every five to ten minutes for two hours with a SpectraMax i3x Multi Mode microplate reader (Molecular Devices, Sunnyvale, CA). The assays were terminated by adding 1% Triton X-100 to the wells and the recorded fluorescence was treated as the maximum for each well. PI uptake was plotted as a percentage of the maximum fluorescence after subtraction of basal fluorescence in control wells treated with propidium iodide without Salmonella.