Mitochondrial ROS Measurement in HUVECs

Mitochondrial ROS production was measured using the fluorogenic dye MitoSOX Red, a mitochondrially targeted derivative of hydroethidine.30 (link) HUVECs on glass cover slips were loaded with MitoSOX Red (5 μmol/L) for 30 minutes. Cells were subsequently treated in duplicate for 20 minutes with equol (100 nmol/L) or vehicle (0.01% DMSO), and fluorescence was detected in 4% paraformaldehyde-fixed cells by confocal microscopy (560/625 nm). Fluorescence images were obtained from a total of 200 cells per cover slip in each of 4 cultures from 4 different donors. In other experiments, cells were pretreated with the cytoskeletal disrupting agent cytochalasin D (2.5 μmol/L) or EGFR tyrosine kinase inhibitor AG-1478 (5 μmol/L) and then stimulated acutely with equol (100 nmol/L) and monitored with MitoSOX Red fluorescence.