Plasma membrane resealing after SLO permeabilization is rapid and requires Ca²⁺ and ATP. (A) Resealing of SLO-permeabilized NRK cells is Ca²⁺ dependent and decreases as toxin concentrations increase. DAPI staining (blue) shows all nuclei; PI staining (red) shows cells that failed to reseal. Bar, 10 μm. (B) FACS of PI staining showing Ca²⁺- and ATP-dependent resealing of cells permeabilized with 200 ng/ml SLO. Dashed lines, no SLO; solid lines, SLO. (C) Ca²⁺ influx into NRK cells loaded with Fluo-3 and exposed to 175 ng/ml SLO stops rapidly under repair conditions but is sustained in ATP-depleted cells. [Ca²⁺]_i transient traces were aligned for graphical representation. See Video 1. (D) FM1-43 influx into HEK-293 cells permeabilized with 200 ng/ml SLO stops rapidly in the presence of Ca²⁺ but is sustained in the absence of Ca²⁺ or ATP. The data represent the mean ± SD (error bars) of intracellular fluorescence intensity in five cells. See Video 2 . RFU, relative fluorescence units.