Fluo-3/AM measurements of intracellular [Ca2+]i

Cardiomyocytes were cultured in confocal culture dishes at a density of 5 × 10⁴ cells/mL and then exposed to 5 μM Fluo-3/AM for 30 min at 37 °C in the dark. Cardiomyocytes were washed twice with Ca²⁺-free PBS to remove extracellular Fluo-3/AM and further incubated in DMEM. Changes in [Ca²⁺]_i were measured by the fluorescence intensity induced by Fluo-3 in cardiomyocytes recorded for 5 min using laser confocal scanning microscopy (Leica, TCS SP5II) with excitation and emission at 488 and 530 nm, respectively. Image Pro Plus 6.0 was used for analysis. In this experiment, the loaded cardiomyocytes were divided into control, H/R, GdCl₃, H/R+AsIV and GdCl₃+AsIV groups.