3.4. Mitochondrial Membrane Potential Detection

3,3′-Dihexyloxacarbocyanine iodide (DiOC6(3)) was purchased from Thermofisher Scientific and applied to monitor mitochondrial membrane potential according to the manufacturer’s instructions (San Jose, CA, USA). Cells were seeded into six-well plates and treated with vehicle or AA for 24 h. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was also applied as a positive control, except that cells were harvested after brief exposure, i.e., 6 h. Cells were trypsinized and washed with PBS two times, followed by DiOC6(3) staining for 20 min in 37 °C water bath. Then cells were washed with PBS for an additional three times, re-suspended with HBSS, and submitted to flow cytometry for analysis (Becton-Dickinson, Franklin Lakes, NJ, USA).