M0393

HUVECs (lot nos. 2818 [black donor] and 2840 [Caucasian donor], 200–05n, Cell Applications, San Diego, CA, USA) were cultured in Medium 199 (M199; 31100–035, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 20% heat-inactivated FBS (12483–020, Gibco or 04–001–1 A, Biological Industries, Beit-Haemek, Israel), 10 µg/L human basic fibroblast growth factor (bFGF; GF-030–3, Austral Biologicals, San Ramon, CA, USA), and 1% penicillin/streptomycin (P/S; 15140–122, Gibco). HUVECs from the fourth to ninth passages were used for experiments in this study. The experiments were conducted using three types of experimental medium (EM): M199 containing 10% heat-inactivated FBS and 1% P/S (EM1), FBS-free M199 (EM2), and a FBS-free M199 with Hank’s salts (M0393, Sigma-Aldrich, St. Louis, MO, USA) (EM3).

MII oocytes for all experimental groups, apart from controls loaded at 37°C, were loaded (for 60 min) at room temperature with the esterified form of the Ca²⁺-sensitive fluorescent probe, Fluo-3AM (5 μM dissolved in 0.1% DMSO plus pluronic acid; Molecular Probes, Eugene, OR, USA). Afterwards, cells were superfused with collection solution (Sigma-Aldrich, M0393) with and without compounds targeting K_ATP channels (described in previous section) and imaged using laser confocal microscopy coupled to an inverted microscope (Leica TCS SP5 II, Milton Keynes, UK) with a ×10 (numerical aperture 1·3) oil-immersion objective lens at 37°C. The intensity of fluorescence of whole oocytes on the equatorial plane was measured. Microscope was calibrated by green calibration slide before each experiment. Intensity of fluorescence was described in arbitrary units (AU) covering a range from 0 to 60 000 AU. Ca²⁺ levels and cell morphology were imaged every 10 min for 2 h using an Argon/UV laser (excitation 488 nm/emission 520 nm). Images were analysed using Leica Application Suite AF Lite software (Leica). The parameters of image acquisition were similar for all examined cells. All compounds were purchased from Sigma-Aldrich. All obtained results were normalized in respect to the intensity of fluorescence at time point 0, that was considered to be 100%.