Mitochondrial Membrane Potential Assay

JC-1 assay was used to detect the depolarization of the mitochondrial membrane potential of the cell after being treated with the selected doses of NLC-Citral for 48 hours using BD MitoScreen Kit (BD Pharmingen, USA). In brief, the cells were seeded in 6 wells plate for 24 hours at a density of 1.8 × 10⁵ cells per well. Then, the cells were treated with various concentrations described in Table 1. This assay was performed based on the manufacturer’s instruction provided with the kit without any modifications. After 48 hours of incubation, the cells were harvested, collected, and incubated with 500 µL of JC-1 stock solution for 15 minutes. Then, it was preceded for analysis by BD FACS Calibur (Becton Dickinson, USA). The result was analyzed using CellQuest 3.3 software. The experiment was performed in triplicates^{81 (link)}.