4.4. Flow-Cytometry Analysis of the Mitochondrial-Membrane Potential

Changes in the mitochondrial-membrane potential can be examined by monitoring the cell fluorescence after double staining with rhodamine 123 (Rh123) and propidium iodide (PI). Rh123 is a membrane-permeable fluorescent cationic dye that is selectively taken up by mitochondria in direct proportion to the MMP (mitochondrial-membrane permeabilization) [47]. B16F10 and A10 cells (4 × 10⁵ cells/well) were seeded on 12-well plates with 2 mL of medium and treated with 0.15 mM H₂O₂ for 24 h and MA at IC_50/4, IC_50/2, IC₅₀ and 2·IC₅₀ (10.6, 21.6, 42.3 and 84.6 μM, respectively) concentrations for 24 h more. Following the treatment, the medium was removed and fresh medium with dihydrorhodamine (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 5 μg/mL of PI. The intensity of fluorescence from Rh123 and PI was determined using an ACS flow cytometer (Coulter Corporation, Hialeah, FL, USA), at the excitation and emission wavelengths of 500 and 536 nm, respectively. The experiments were performed three times with two replicates per assay.