4.6. ROS Detection Assays

The measurement of intracellular ROS was determined by the Cellular Reactive Oxygen Species Detection Assay Kit (Abcam) as per the manufacturer’s instructions. To detect ROS levels, the cell-permeant, non-fluorescent 2′,7′-dichlorofluorescein diacetate (DCFDA) is oxidized by hydroxyl, peroxyl or other ROS to fluorescent DCF. Cells were stained with DCFDA or assay buffer as a negative control, and then treated with ZnPP, ketoconazole, or vehicle control in the dark for the indicated times. Fluorescence was detected with excitation and emission wavelengths at 485 and 535 nm, respectively. Fluorescence DCF values were normalized to the corresponding cell viability data.

The superoxide generation in the mitochondria was further visualized by DHE (Sigma-Aldrich) staining. DHE freely enters cells and is oxidized by superoxide to ethidium bromide which emits red fluorescence. Cells were treated with ZnPP, ketoconazole, or vehicle control for 24 h, and then fixed and stained with DHE for 30 min. Nuclear counterstaining was performed using 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Samples were examined under a fluorescence microscope.