2.7. Assay of Mitochondrial Trans-Membrane Potential (JC-1 Staining)

Mitochondrial trans-membrane potential was measured using the fluorescent probe JC-1, which produces green fluorescence in the cytoplasm and red to orange fluorescence when concentrated in mitochondria that are metabolically active and so maintain a negative internal potential [53]. JC-1 staining was performed according to the manufacturers’ protocol (Sigma, St. Luis, MI, USA). Cells treated for 6 and 12 h with 24 h IC₅₀ of the complex in 12-well plates were incubated for 30 min with JC-1 (2 µg/mL) prepared in the culture medium. The adherent cell layer was then washed with PBS and examined in the fluorescent microscope using a UV filter (450–490 nM). Three hundred cells per sample were counted, in duplicate, for each dose- and time point. Orange-red fluorescence of cells indicated intact mitochondrial membrane whereas green fluorescence indicated loss of mitochondrial membrane integrity. These specific fluorescent patterns were indicative of live and dead cells, respectively (if dead, apoptotic or necrotic).