Mitochondrial ROS Imaging in Cells

Mitochondrial ROS production was evaluated using the mitochondrial-targeted dye Mitotracker CM-H2XRos [55 (link)] (Invitrogen Life Technologies, Carlsbad, CA, USA). RA-differentiated cells were cultured on glass coverslip on 6-well plates (1 × 10⁵ cells/well) and, after being treated, cells were loaded with 300 nM of the dye for 30 min in the dark at 37 °C. For live cell imaging of mitochondrial ROS, measurements were conducted on the confocal microscope Zeiss 510 LSM (Carl Zeiss, Milan, Italy). CM-H2XRos was excited with 561 nm laser, and emission was detected above 580 nm and recorded for 120 s. The analysis of red fluorescence increase was performed off-line after images acquisition. Fluorescence values were reported as percentage of the control.