Mitochondrial Membrane Potential Assay

Measurement of mitochondrial membrane potential was performed using a JC-10 Assay Kit (Abcam, Cambridge, UK) as previously described (Przepiórska et al. 2023 (link)). JC-10 transpires into mitochondria and polymerizes there in cells with proper mitochondrial membrane potential, forming aggregates with characteristic red fluorescence. Low mitochondrial membrane potential results in a lack of JC-10 transport into mitochondria, leading to JC-10 green fluorescent monomer deposition in the cytoplasm. The cell culture medium was replaced with phosphate-buffered saline (PBS) (Biomed, Poland). After a 15-min incubation, the JC-10 dye solution was added, and the cells were incubated with the reagent for another 30 min. Then, Assay Buffer B was added, and immediate measurements at Ex/Em = 490/525 nm (green fluorescence) and 540/590 (red fluorescence) were performed using the Infinite M200PRO microplate reader (Tecan, Switzerland) and Tecan i-control software. The next step of the analysis was a calculation of the red to green fluorescence ratio. The obtained values were normalized to those of vehicle-treated cells and are expressed as a percent of the control ± SEM.