Mitotracker probes and mitochondrial membrane potential

Isolated Mitochondria Infusion Mitigates Ischemia-Reperfusion Injury of the Liver in Rats: Reply Reply In our previous study (1), we showed that infusion of freshly isolated mitochondria via spleen significantly mitigated ischemia-reperfusion injury of the liver. We used JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) staining and flow cytometry analysis to examine the membrane potential of the isolated mitochondria (Δψm) before the infusion. Besides, we used MitoTracker Orange CMTMRos (5 μmol/L; Invitrogen, Carlsbad, Calif) to stain the isolated mitochondria and track their biodistribution in the liver. After 240 min of reperfusion, we observed the MitoTracker Orange–labeled mitochondria distributed among the cryosectioned liver parenchyma. We proposed that the infused mitochondria still maintained their membrane potential after infusion. Thanks to Dr. Lemasters et al., they reviewed the chemistry and questioned that MitoTracker Orange CMTMRos would keep binding to mitochondrial proteins irrespective the change in Δψm. To solve the problem, we should also counterstain the infused mitochondria with the mitochondria-specific dye MitoFluor Green (Invitrogen), which would stain mitochondria with and without intact membrane potentials. After deducing the MitoTracker Orange–stained mitochondria, we could then estimate the ratio of mitochondria maintaining intact Δψm. According to the report of McCully et al. (2), they used the MitoFluor Green counterstaining and estimated that less than 0.05% of injected mitochondria were not viable.