ROS and mitochondrial superoxide production were determined using a fluorescent dye chloromethyl-2′,7′-dichlorofluorescein diacetate (H2DCF-DA, Sigma, 35845) and a specific mitochondrial superoxide indicator MitoSOX Red (Thermo Fisher, {"type":"entrez-nucleotide","attrs":{"text":"M36008","term_id":"214108","term_text":"M36008"}}M36008), respectively. Briefly, cells with specified treatments were washed with PBS and incubated with 5 μM H2DCF-DA (or 10 μM MitoSOX) at 37 °C for 30 min to load the fluorescent dye. Afterward, cells were washed twice with PBS, and trypsinized, centrifuged, resuspended in 500 μl PBS. Cells were kept in the dark until analysis by flow cytometry (Accuri C6, BD Biosciences).
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