Annexin-containing EVs are shed from the repair scab after plasma membrane damage. (A) Representative confocal micrographs of FM1-43 infiltration are shown. Image times are relative to the first image taken after ablation. Large arrows in panes I and II indicate the site of ablation. Small arrows in panes II and III indicate the staining of intracellular compartments in an ablated (lower arrows) and a non-ablated cell (upper arrows). Large arrows in panes III and IV indicate EVs. Scale bars: 5 μm. (B) Quantification of mRNA-seq reads mapping to each annexin paralog is shown. (C) Representative confocal micrographs of ANXA2-mScarlet recruitment after ablation are shown. Image times are relative to the first image taken after ablation. White arrows in panes I and II indicate the site of ablation. Arrow in pane III indicates an EV. Scale bars: 5 μm. (D) Sytox Green staining over time in the absence of extracellular Ca²⁺ after cells pretreated with the indicated concentration of SLO was rapidly heated from 4 to 37°C. Error bars indicate three experimental replicates. (E) Membrane-protected luminescence in medium fractions at indicated time points after ANXA2-Nluc or CD63-Nluc cells pretreated with 200 ng/μl SLO were rapidly heated from 4 to 37°C is shown. (F) EV production index from ANXA2-Nluc or CD63-Nluc cells treated with increasing concentrations of SLO is shown. Error bars indicate three experimental replicates.