2.7. Determination of ROS Production

Dihydroethidium (DHE, BestBio, China) staining was used to detect ROS levels in heart tissue. Fresh mouse heart tissue samples were embedded in an OCT compound (Thermo Fisher). Cryopreserved sections were then loaded with 500 μM DHE following the manufacturer's instructions. Oxidized DHE was excited at 543 nm, and emission was collected with a LP 560 nm filter using the NIKON T12-E fluorescence microscope.

Intracellular ROS levels in H9c2 cells were determined by measuring the oxidative conversion of cell-permeable 2′,7′-dichlorofluorescein diacetate (DCFH-DA) to fluorescent dichlorofluorescein (DCF). The cells were washed with D-Hank's balanced salt solution (HBSS) and incubated with DCFH-DA at 37°C for 20 min. Then, fluorescence was detected by flow cytometry at Ex (λ) 488 nm, Em (λ) 525 nm (Beckman Coulter), which would collect and analyze ten thousand cells in each flow cytometric assay.