2.7. Intracellular ROS and Mitochondrial ROS Detection

Intracellular ROS levels were determined by DHE and DCFH-DA staining. The pretreated H9c2 cells were incubated with 10 μM DHE solution [32] at 37°C for 30 min without light. Then, DHE was removed by washing with PBS twice. The cells were imaged on a fluorescent microscope camera (Olympus IX71, Olympus Corporation, Tokyo, Japan). And the fluorescent intensity of DHE was analyzed with Image J software (National Institutes of Health, Bethesda, MD, USA). For DCFH-DA staining, after treated with NOX2 inhibitor peptide (GP91ds-tat, 5 μM [33]), scramble GP91ds-tat (scrGP91ds-tat, 5 μM), NAD(P)H oxidase inhibitor (apocynin, 0.3 mM [34]), and mitochondrial ROS scavenger (Mito-TEMPO, 10 μM [33]), H9c2 cells were incubated with 10 μM DCFH-DA [35] at 37°C for 30 min without light. Then, DCF-loaded cells were washed and analyzed by flow cytometry (wavelength 488/538 nm).

For mitochondrial ROS detection, H9c2 cells were loaded with MitoSOX (5 μM [36, 37]) at 37°C for 30 min followed by washing twice with HBSS buffer containing Ca²⁺/Mg²⁺. Subsequently, cells were aliquoted at a density of 5‐10 × 10⁶ cells in a sterile FACS tube and analyzed by flow cytometry (emission wavelength 580 nm).