*4.8. Mitochondrial Membrane Potential (MMP) Assessment for Localization of Cytochrome-c*

The mitochondria-specific fluorescence dye, namely, 5,5',6,6'-tetrachloro-1,1'3,3'- tetraethyl benzamidazol-carbocyanine iodide (JC-1) (JC-1 MMP assay kit, Abcam), was used to determine the MMP according to the method previously described [55]. HepG2 cells were seeded in 12-well plates and treated with EO (10 and 25 μg/mL) for 24 h. JC-1 working solution was added and incubated at 37 ◦C for 20 min. Treated cells were washed twice with PBS, replaced with fresh DMEM medium, and captured on a phase contrast inverted fluorescence microscope 200X (Leica 3000 fluorescence microscope). Mitochondrial membrane potentials were monitored by the ratio of red and green fluorescence intensity.
