Measuring Cellular Oxidative Stress

H9c2 cells seeded on glass coverslips were loaded with the ROS-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; Invitrogen, CA, USA; 2.5 μmol L⁻¹) or the mitochondrial ${\mathrm{O}}_2^{\cdot -}$ -specific fluorescent probe MitoSOX (Invitrogen; 3 μmol L⁻¹) - dissolved in 0.1% DMSO and Pluronic acid F-127 (0.01% w/v) – which were added to cell culture media for 15 min at 37 °C, as described^{18 (link)}. The cells were fixed in 2% buffered paraformaldehyde for 10 min at room temperature and the H₂DCFDA and MitoSOX fluorescence analysed using a Leica TCS SP5 confocal scanning microscope equipped with an argon laser source (excitation λ 488 nm or 543 nm, respectively) and a x63 oil immersion objective. ROS and mitochondrial ${\mathrm{O}}_2^{\cdot -}$ generation were also monitored by flow cytometry^{54 (link)}: briefly, single-cell suspensions were incubated with H₂DCFDA (1 μmol L⁻¹) or MitoSOX (0.5 μmol L⁻¹) for 15 min at 37 °C and immediately analysed using a FACSCanto flow cytometer (Becton–Dickinson). Data were analyzed using FACSDiva software (Becton–Dickinson).