PM wounding by SLO increases endocytosis. (A) Confocal fluorescence microscopy images of Texas red–dextran and cholera toxin B subunit (CTB) in B cells. Cells were stained with Alexa Fluor (AF) 488–CTB (3 µg/ml) at 4°C and incubated with or without SLO at 37°C in the presence of dextran (2.5 mg/ml) for 3 min. Cells were then washed and stained with AF405 goat anti–mouse IgG to label surface BCR at 4°C. Bar, 2.5 µm. (B) Percentage (± SD) of B cells (treated or not with SLO or SM) showing internalized dextran, determined by visual inspection of confocal images from three independent experiments. (C) Correlation coefficients of dextran and CTB staining in the presence or absence of SLO, determined by confocal fluorescence microscopy. Shown is the mean (± SD) of three independent experiments. (D and E) Quantification of CTB endocytosis by flow cytometry. B cells were labeled with AF488-CTB (1 µg/ml) at 4°C and treated with SLO at 37°C for 3 min. The surface CTB was quenched with anti-AF488 antibodies at 4°C before and after SLO treatment. The MFI of CTB was quantified by flow cytometry. Shown are a representative histogram (D) and the percentage (± SD) of internalized CTB from three independent experiments (E). (F and G) TEM analysis of CTB endocytosis. B cells were incubated at 4°C with biotin-CTB (2 µg/ml) followed by gold-streptavidin (10 nm, 2 µg/ml), and then treated with SLO for 1 and 5 min at 37°C. Shown are representative images (F) and the mean percentage (± SD) of internalized CTB gold particles from >16 randomly selected cell profiles per condition from two independent experiments (G). Bar, 100 nm. **, P < 0.005; ****, P < 0.0001.