2.5. Mitochondrial Oxidative Stress Measurement by Mitosox Staining Method

The H9C2 cardiac cells were cultured at a density of 1 × 10⁵/cells/ml. The cells were treated with 35 mM glucose for 24 h in the CO₂ incubator at 37°C. Mitosox (Thermo Fisher Scientific, USA) was added to the final concentration at 5 μM in the medium according to the manufacturer's recommendation. After 10 min, the cells were washed 2 times with Hank's Buffered Salt Solution (Gibco, USA) containing calcium and magnesium before measurement. The fluorescent image and intensity of Mitosox were measured at 510 nm (excitation wavelength) and 580 nm (emission wavelength) by fluorescent microscopy (FV-1200MPE SHARE, Olympus Japan). The ratio of cell fluorescent intensities of Mitosox to the fluorescent beads was calculated.