Mitochondrial Membrane Potential Assay

The mitochondrial membrane potential assay can be used to evaluate cell function and health; thus, cationic carbocyanine dye (JC-1) staining cells were used [35 (link)]. Thus, ConA-stimulated T lymphocytes exposed to IC₅₀ doses of P. alata extract and polyphenols were washed with PBS 0.1 M (pH 7.4). After centrifuging at 300× g for 5 min at room temperature, the cells were labelled with 1 µg of JC-1 dye (Santa Cruz Biotechnology, Dallas, TX, USA), diluted in 100 µL of PBS and incubated at 37 °C for 30 min. The cells were then washed with PBS, centrifuged for 5 min at 300× g and analyzed immediately by flow cytometry. T cells were submitted to valinomycin (Cayman Chemical, Ann Arbor, MI, USA) as a positive assay control at 100 µM. The results are expressed as the percentage of depolarized (green fluorescent) and polarized (orange–red fluorescent) T lymphocyte mitochondrial membrane potential.