Movie 1.

Aerolysin triggers Ca²⁺ flux. HeLa cells were labeled with Fluo-4-AM (green) for 30 min in DMEM. The media was replaced with imaging buffer (2 μg/mL TOPRO3 (blue), 25 mM HEPES, pH 7.4, and 2 mM CaCl2 supplemented RPMI). Cells were imaged by confocal microscopy at 1-3 frames/sec for ∼45 min at 37°C. Sublytic (A) SLO (250 HU/mL), or (B) aerolysin (62 HU/mL) was added 5 min after imaging started. Triton-X-100 was added just prior to the end of imaging as a positive control for cell permeabilization. Images were bleach-corrected by histogram matching. Time displays min post toxin challenge. Representative Ca2+-flux is shown by arrowheads. Scale bar = 10 μm.