Quantifying Mitochondrial and Cellular ROS

Mitochondrial ROS and intracellular ROS in BUMPT cells were determined by using Mito-SOX Red (Thermo Fisher Scientific, M36008) and by CM-DCFDA (Invitrogen, C6827), respectively. ROS in kidney tissues was measured by dihydroethidium (Thermo Fisher Scientific, D11347) staining as previously described (26 (link)). Briefly, after harvesting, kidneys were snap frozen by liquid nitrogen. 20 mm sections of the kidneys were incubated with 10 mM DHE in a humidified and dark chamber at 37°C for 30 min and then counterstained with DAPI (Sigma-Aldrich, D9542). Images of the above staining were captured with fluorescence microscopy. For quantification, the fluorescence intensity in the nuclei of proximal tubular cells within 10 random optical sections was determined with ImageJ software. At least 10 randomly selected fields per mouse and four mice for each group were examined.