Application suite af lite software

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%.

Stable KG1a cells were transfected with GFP-PKCα and plated on 25 μg/μl of fibronectin overnight. Cells were imaged using the Leica SP8 System using a 63X water objective equipped with an objective heater which maintained samples as 34 °C throughout imaging. The excitation light source was a white-light laser system set at 488 nm (GFP) and 561 nm (mCherry). Fluorescence from the 488 nm channel was collected using a HyD1 detector and fluorescence from the 561 nm channel was collected using the HyD SMD2 in standard mode. Photobleaching was performed at 100% 561 nm laser power for 5 frames. GFP and mCherry levels in cells outside of the field of bleaching demonstrate that inherent photobleaching did not play a significant role in reducing GFP or mCherry fluorescence over the course of imaging (Supplemental Fig. 4). FRET efficiencies were calculated using the formula: Efficiency = (Donor_post-bleach − Donor_pre-bleach)/Donor_post-bleach where D is the fluorescence intensity in a plasma membrane region of interest of fixed shape and size (3 × 7 ellipse). Analysis was performed using the Leica Application Suite AF Lite software.