Assessing Mitochondrial Membrane Potential

Alterations in mitochondrial membrane potential (MMP) were determined by using the fluorescent dye Rh-123 staining method as reported earlier [21 (link), 22 (link)]. Briefly, cells were seeded in a 6-well plate and treated with different concentrations of HMF for 24 h. Cells were washed with PBS and stained with 1 μg/ml of Rh-123 at 37°C for 30 min in the dark. After washing with PBS, fluorescence intensity of cells was analyzed under a fluorescence microscope (Nikon Eclipse TS100 Epi-fluorescence microscope, Japan). Mean fluorescence intensity was quantified using ImageJ software after background staining correction.