Mitochondrial-membrane potential

Changes in the mitochondrial-membrane potential can be examined by monitoring the cell fluorescence after double staining with Rh123 (rhodamine 123) and PI. Rh123 is a membrane-permeable fluorescent cationic dye that is selectively taken up by mitochondria directly proportional to the MMP (mitochondrial membrane permeabilization). In the same way as in the cell cycle assays, 4 × 10⁵ cells/well were plated on 6-well plates with 2 mL of medium and treated with cytotoxic activity compounds for 4 h at IC₅₀ and IC₈₀ concentrations. After treatment, the medium was removed and a fresh medium with DHR, at a final concentration of 5 μg/mL, was added. After 30 min of incubation, the medium was removed and the cells were washed and resuspended in PBS with 5μg/mL of PI. The intensity of fluorescence from Rh123 and PI was determined using a FACScan flow cytometer (Coulter Corporation, Hialeah, FL, USA), using excitation and emission wavelengths of 500 and 536 nm, respectively.