2.7. Mitochondrial Activity

Mitochondrial activity expressed through the mitochondrial membrane potential (ΔΨm) was evaluated with a JC-1 Mitochondrial Membrane Potential Assay kit (Cayman Chemical, Ann Arbor, MI, USA). The JC-1 fluorescent dye (5.5′,6.6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) was dissolved in PBS to constitute the working solution (5 μmol/L JC-1), 5 μL of which was mixed with 1 × 10⁶ cells adjusted to 100 μL and incubated at 37 °C for 30 min. Subsequently, each sample was centrifuged (150× g, 5 min, 20 °C) and washed twice with a JC-1 washing buffer. Finally, the specimens were transferred to a dark 96-well plate and analyzed using a GloMax-Multi + fluorometer (Promega, Madison, WI, USA). The resulting ΔΨm is expressed as the green/red ratio (JC-1 complexes/JC-1 monomers) [19].