Fig. 7.

Ca²⁺ influx occurs prior to total membrane permeabilization in pyroptosis. a–d B6^Nlrp1b+ BMDMs preloaded with the cell-permeant Ca²⁺ indicator Fluo4 were imaged after stimulation with LeTx (a, b) or FlaTox (c, d) in culture media containing PI. Confocal images were acquired every 1.5 min. Graphs show the percentage of mean fluorescence intensity (MFI) calculated as described in the Methods section, and values represent the mean ± SD of individual cells imaged in 4 independent experiments (LeTx, n = 24; FlaTox n = 23). Single-cell plots are shown in Supplemental Figure 10. Fluorescent micrographs show the maximum intensity projection of a representative cell. In all panels, time point zero indicates the first detection of PI. All scale bars, 10 µm