Intracellular calcium measurement

PC3 cells were grown on 25-mm diameter glass coverslips and kept in DMEM F12 medium for 24 h. Subsequently, the cells were washed with PBS and loaded with 2 µM Fluor-4 acetoxymethyl ester (Fluo4/AM; Thermo Fisher Scientific, Inc.) at room temperature for 15 min. Subsequently, the cells were maintained in Hank's Balanced Salt Solution at a final volume of 500 µl containing 142 mM NaCl, 5.6 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 0.34 mM Na₂HPO₄, 0.44 mM KH₂PO₄, 4.2 mM NaHCO₃, 10 mM HEPES and 5.6 mM glucose. On the one hand, 100 nM ET-1 was added at 60 sec after reaction was initiated with Fluo4/AM, at room temperature, to cells previously incubated with DMSO or with selective antagonists for endothelin receptors, at 37°C for 30 min. In all experiments DMSO (0.1%) was used. On the other hand, 17 mM carbachol (positive control) was added at 245 sec after reaction was initiated with Fluo4AM, at room temperature, to cells preincubated with BQ123 at 37°C for 30 min. The kinetics recording was carried out for 300 sec and the fluorescence intensity was measured in an AxioCam MRm (Carl Zeiss AG) coupled to a Carl Zeiss AG inverted fluorescence microscope AxioVert. A1 equipped for epifluorescence and the ImageJ 1.51w software (National Institutes of Health) was used.