For port A, I injected 5 mM ADP rather than directly adding it to the wells, but I don’t believe this is the main reason for the problem, especially since the issue also persists when only oligomycin (40 µM, port B), FCCP (80 µM, port C), and Rot/AA (20 µM, port D) were injected. According to my Bradford assay, I loaded 5 µg of isolated mitochondria into wells.