Fig. 2. (A) Menadione, but not ACh, CCK or thapsigargin induce apoptosis in a Ca²⁺-dependent manner. Annexin V FITC ApoAlert Kit detects apoptosis in pancreatic acinar cells after incubation with menadione. Each column represents the intensity of Annexin V FITC fluorescence of every propidium iodide negative cell in a field of view (control cells, cells after 30 minutes incubation with 5 pM CCK or 25 nM ACh or 100 nM ACh or 1 µM thapsigargin for 30 minutes or for 3 hours, 250 µM ZVADfmk + menadione, BAPTA + menadione, 50 µM Bongkrekic acid + menadione or menadione alone for 15 minutes and for 30 minutes) measured in the same conditions using the confocal microscope. a.u, arbitrary units. (B) Pre-treatment of cells with 25 µM BAPTA AM prevents cytosolic Ca²⁺ spikes induced by menadione. Cells were loaded with Fura Red AM. (C) Cell death as measured by 2.5 µg/ml propidium iodide staining of the nucleus 6 hours after addition of 20 µM menadione or vehicle control. (D,E) Nuclear apoptosis in cells 3 hours after addition of 20 µM menadione (E) or controls immediately after isolation (D) is shown. Nuclei were stained with 8 µg/ml Hoechst 33342 for 15 minutes and imaged with a Leica SP2 confocal microscope. a.u., arbitrary units.