The microscopic observation of intracellular ROS and mitochondrial ROS was performed by the oxidative conversion of cell permeable DCFH-DA to fluorescent 2′, 7′-dichlorofluorescein (DCF) and MitoSOX (M36008, Invitrogen; Thermo Fisher Scientific, Inc.). CHs were divided into the control, TBHP, TBHP + GYY4137 and TBHP + NAC group, then stained with 10 μM DCFH-DA solution or 5 μM MitoSOX at 37°C for 20 min in the dark. The mean fluorescence intensity (MFI) of DCF and MitoSOX from 4 random fields was observed using an imaging system (BX50-FLA; Olympus Corporation) and analyzed using ImagePro Plus software (version 6.0, Media Cybernetics).

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