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Flow cytometry analysis for mitochondrial ROS
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Mitochondrial superoxide was measured following the same protocol as described in Xiong et al.14 Briefly, cells cultured on 60-mm dishes were incubated with fresh complete medium containing 5 μM MitoSOX Red (Life Technologies, M36008) at 37 °C. After 30 min, stained cells were harvested by trypsin digestion and rinsed twice with PBS. Single cell suspensions in 400 ml PBS were prepared for FACS analysis (FACS Canto II; BD). MitoSOX Red was excited by a laser at 488 nm, and the data was collected at 582 ± 21 nm.
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