Mitochondrial membrane potential assay

The mitochondrial membrane potential was determined using the Mito-ID membrane Potential Cytotoxicity Kit (Enzo Life Sciences, Farmingdale, NY, USA). In energized inner membrane, the mitochondria produced an orange fluorescence signal. If cells exhibit a shift from orange to green fluorescence: mitochondrial function becomes increasingly compromised. Freshly isolated PBMCs were stained with mito-ID membrane potential dye loading solution in black, clear-bottom 96-well tissue culture dishes for 30 min. After incubation, the resulting fluorescence was visualized by fluorescent microscopy (Evos Digital microscope, Evos, Hatifiled, PA, USA).