Reactive oxygen species (ROS) measurement

Intracellular superoxide anions were measured using the dihydroethidium (DHE) fluorescence probe. H9c2 cells were incubated in a light-impermeable chamber at 37 °C for 30min after application of 10 µmol·L⁻¹ DHE (Life Technology, USA) and were then cultured with 5 µg·mL⁻¹ 4',6-diamidino-2-phenylindole (DAPI, Sigma, USA) for 5 min. The images of H9c2 cardiomyocytes were captured and analyzed immediately under an inverted fluorescence microscopy (Olympus BX51, Japan). The intracellular ROS level was quantified using a redox-sensitive dye carboxy 2'7'-dichlorodihydrofluorescein diacetate (H₂DCF-DA) obtained from Molecular Probes (Life Technology, USA). H₂DCF-DA is non-fluorescent, after meeting oxidation by ROS it converts to fluorescent marker 2'7'-dichlorofluorescein (DCF) which emits green fluorescence. In brief, H9c2 cells were treated with 1 µmol·L⁻¹ H₂DCF-DA for 30 min in a conventional incubator (37 °C, 5% CO₂). Cardiomyocytes were rinsed with phosphate buffer saline (PBS) and DCF fluorescence intensity was measured using a fluorescence plate reader (Molecular Devices, Sunnyvale, CA) with 480 nm excitation and 530 nm emission filters (17).