2.8. Detection of MMP

When the mitochondrial membrane potential (MMP) is relatively elevated, JC-1 forms a polymer in the mitochondrial matrix, resulting in the emission of red fluorescence. Conversely, when the MMP decreases, JC-1 is unable to aggregate within the matrix, resulting in green fluorescence production [20]. Hence, the proportion of red and green fluorescence is frequently employed to assess alterations in MMP. The procedures for cell treatment were identical to those described in Section 2.7. Following the completion of the treatment, cells were harvested and suspended in culture media at a concentration of 1 × 10⁵ cells/mL before being subjected to incubation with JC-1 staining solution (5 μg/mL) at 37 °C for a duration of 20 min. After applying staining, the FACS flow cytometry technique was employed to analyze the intensity of the red fluorescence emitted by the JC-1 aggregates. This was accomplished by utilizing an excitation wavelength of 525 nm and an emission wavelength of 590 nm. Additionally, the study employed a 490 nm excitation wavelength and a 530 nm emission wavelength to measure the green intensity of JC-1 monomers. The FlowJo software version 9.3.2 (TOMY Digital Biology, Tokyo, Japan) was utilized to calculate the relative ratio between the red and green fluorescent intensities, enabling us to determine the extent of mitochondrial depolarization.