Mitoflash activity mitigates in state III versus state II/IV respiration. (A) Experimental design. In the presence of Complex I, II, or IV substrates, 200 µM ADP was added to switch respiring mitochondria from state II/IV to state III. Oligomycin (Oligo, 5 µM) was used to inhibit ATP synthase. The black arrow shows the electron transfer pathway. (B–D) Effects of ADP and oligomycin on mitoflash frequency supported by Complex I substrate (B), Complex II substrate (C), or Complex IV substrate (D). Data are mean ± SEM, n = 11–18 (B), 12–17 (C), and 13–16 (D) image series from three mice. ***, P < 0.001 ADP group versus respective control group.