Mitochondrial ROS Measurement in Endothelial Cells

Mitochondrial reactive oxygen species generation was measured using a mitochondrial targeted fluorogenic probe MitoSOX™ Red [34 (link)], and we previously confirmed that MitoSOX fluorescence in endothelial cells is attenuated by scavenging superoxide [28 (link),35 (link)]. bEnd.3 cells seeded in black-walled, clear-bottomed 96-well plates were cultured under 18 or 5 kPa O₂ for 5 d and then incubated in serum-free DMEM in the absence or presence of rotenone (1 μM, complex 1 inhibitor) or l-NAME (100 μM, eNOS inhibitor). Cells were exposed to hypoxia (1 kPa O₂, 1 h) and loaded with MitoSOX™ Red (5 μM, Invitrogen) for 5 min before the start of reoxygenation under 18 or 5 kPa O₂, respectively. Cells were washed twice with ice-cold PBS and fixed with 4% paraformaldehyde for 10 min before staining nuclei with DAPI (2 μg/ml, Sigma). MitoSOX™ Red fluorescence (Ex 545 nm/Em 602 nm) was detected using a Nikon Diaphot microscope, with images captured using an ORCA-03G (Hamamatsu, Japan) camera with 0.89 s exposure. Fluorescence quantification was conducted using image analysis software (ImageJ, NIH, USA), measuring the integrated intensity of fluorescence, area of field of view and mean grey value.