Rhodzin 3

The free zinc (Zn²⁺) concentration in the mitochondria of H9c2 cells was assessed with RhodZin-3 according to the manufacturer's instructions (MolecularProbe) and previous studies [29 (link), 30 (link)]. After delayed preconditioning induced by diazoxide or 48 hours after siRNA transfection, H9c2 cells were cultured in a specific temperature-controlled culture dish. The dish was coloaded with both 5 μM RhodZin-3 plus 0.02% pluronic acid (MolecularProbe) and 100 nM Mito-Tracker Green (Beyotime, China) in fresh medium without FBS for 30 min at 37°C in the dark in a humidified 5% CO₂ + 95% air atmosphere. After 3 washes with phosphate-buffered saline, the cells were incubated for another 30 min for deesterification and then mounted on the stage of confocal microscope in a humidified 5% CO₂ + 95% air atmosphere (Leica, Germany). The temperature was maintained at 37°C with a Delta T Open Dish System (Bioptechs, Butler, PA), and the fluorescence intensity was measured as described above with Image Pro-Plus 5.1.