Tcs sp5 2 epifluorescence confocal microscope

H9c2 cells were grown on a coverslip glass, and differentiation conditions were initiated 24 h after being seeded. Prior to loading with fluorescent dyes, the cells were preincubated for 3 min in Tyrode solution (120 mM NaCl; 5 mM KCl; 0.4 mM NaH2PO4; 0.5 mM MgCl2; 25 mM HEPES; 1 mM CaCl2; 0.5 mM glucose, pH 7.4) and loaded with 10 µM Fluo-4 for 45 min at 37 °C in dark conditions. Afterward, coverslips were rinsed with fluorophore-free Tyrode and mounted in a perfusion chamber. Ca2+ transients were evoked by perfusion of caffeine (20 mM, 58-08-2) followed by perfusion of vasopressin (100 nM, V9879) [12 ]. Fluorescent images were acquired using a Leica TCS SP5 II epifluorescence confocal microscope (Leica Microsystems, Wetzlar Germany), using a 40× oil immersion objective, and analyzed by ImageJ software (http://imagej.nih.gov/ij/, NIH, Bethesda, MD, USA). Fluorescence data is shown as ΔF/F0, where F0 is the average fluorescence intensity before caffeine perfusion.

The cellular area and counting of polynucleated cells following cell-differentiation protocol were performed in live cells by fluorescence using calcein-AM to stain against cell cytoplasm and DRAQ to stain nuclei. Briefly, live cells seeded in coverslips were washed with Tyrode 1x (120 mM NaCl; 5 mM KCl; 0.4 mM NaH2PO4; 0.5 mM MgCl2; 25 mM HEPES; 0.5 mM glucose, pH 7.4), and then incubated at 37 °C in a 5% CO2 and 95% air-humidified atmosphere for 30 min, with a 1 µM calcein (Invitrogen, MA, USA). Afterward, coverslips were mounted in a perifusion chamber, and immediately prior to acquiring fluorescent images, cells were incubated for 5 min with nuclei-staining probe Draq5™ (10 μM, Thermofisher Scientific, MA, USA). Fluorescent Images were acquired using a Leica TCS SP5 II epifluorescence confocal microscope (Leica Microsystems, Wetzlar Germany), using a 40× oil immersion objective. The fluorescence calcein was assessed using an Ar laser at 488 nm, and emission was acquired at 517 nm with a bandwidth of 20 nm; DRAQ5 was assessed using a He-Ne laser at 633 nm (excitation), and emission was acquired at 733 nm with a bandwidth of 66 nm. The area of positive calcein fluorescence (surrogate of cell area), as well as the number of nuclei, was determined for each cell. Image analysis was performed using ImageJ software (1.50a; http://imagej.nih.gov/ij/, NIH, Bethesda, MD, USA).