Intracellular Reactive Oxygen Species (ROS) Measurement

Intracellular general ROS level was assessed by the fluorescent probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) (ab113851, Abcam, United States). Cells were incubated with 20 μm DCFH-DA at 37°C for 30 min in dark. The fluorescent images of DCFH-DA staining were acquired by using a laser scanning confocal microscopy at an excitation wavelength of 488 nm and an emission wavelength of 525 nm (LSM510, Carl Zeiss, Germany). The average fluorescence intensity was analyzed by using an image analysis system to identify the general ROS production (Image J, National Institutes of Health, United States).

Additionally, mitochondrial superoxide production was evaluated by MitoSOX™ Red ({"type":"entrez-nucleotide","attrs":{"text":"M36008","term_id":"214108","term_text":"M36008"}}M36008, Invitrogen/Molecular Probes, United States). Cells were loaded with 5 μm MitoSOX™ Red at 37°C for 30 min in dark, and were imaged by a laser scanning confocal at an excitation wavelength of 510 nm and an emission wavelength of 580 nm (LSM510, Carl Zeiss, Germany). MitoSOX Red fluorescence was analyzed by an image analysis system to identify the mean mitochondrial superoxide production (Image J, National Institutes of Health, United States).