Assessing Oxidative Stress in H9c2 Cardiomyocytes

H9c2 cardiomyocytes were donated by Dr. He from Hubei university of medicine [17 (link)]. Cells were cultured in high glucose dulbecco’s modified eagle medium (DMEM) supplemented with 10% FBS (Gibco, C11995500), 100 IU/mL of penicillin and 100 μg/mL of streptomycin, and incubated in 95% air, 5% CO₂. The detection of reactive oxygen species (ROS) and ∆Ψm in H9c2 cells was carried out according to the specifications of ROS detection kit (Beyotime, S0033) and mitochondrial membrane potential detection kit (Solarbio, CA1310). For the determination of ROS, 1 × 10⁵/well H9c2 cells were first cultured under normal conditions (control group), O₂ 1% or O₂ 1% + QLQX, and then incubated with reactive oxygen species sensitive dye 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) solution at 37 °C for 20 min. In order to measure ∆Ψm, cells were treated as described above and then incubated with 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) staining solution (5 mg/ml) at 37 °C for 20 min. After staining, cells were washed twice with JC-1 staining buffer and detected by fluorescence microscope (Olympus FV3000RS). Fluorescence is measured at excitation/emission 485/580 nm (red) and then at excitation/emission 485/530 nm (green). The results were analyzed with Image-Pro Plus software.