Measurement of Mitochondrial Membrane Potential

JC-1 mitochondrial membrane potential assay kit (Abcam Australia Pty. Ltd.) was used to measure mitochondrial membrane potential. JC-1 is a cationic fluorescent dye that accumulates within the mitochondria in a membrane potential-dependent manner. After 24-h exposure to either BSA, PA, or LA, JC-1 was directly administered directly to cell culture media at a final concentration of 10 μM and cells incubated for 10 min at 37°C in the dark. Cells were then washed three times with PBS and JC-1 red fluorescence recorded at 550/615 (excitation/emission) and green fluorescence at 489/535 (excitation/emission) using Victor3 V 1420 Multilabel Counter (PerkinElmer). Finally, the ratio of red to green fluorescence was calculated to quantify mitochondrial membrane potential. The green fluorescence quantified at 489/535 (excitation/emission) is directly proportional to mitochondrial membrane potential, whereas red fluorescence, detected at 550/615 (excitation/emission), represents JC-1 aggregates that accumulate within the mitochondria when membrane potential exceeds −240 mV (44). In light of this, calculating the ratio between red vs. green fluorescence allows the quantification of mitochondrial membrane potential independently of the number of mitochondria assessed. After fluorescence quantification, cells were imaged using the EVOS imaging system to obtain representative images (Thermo Fisher Scientific).