Figure 1: Onset of the MPT after I/R in primary rodent hepatocytes. After 4 hours of simulated ischemia, hepatocytes were reperfused and confocal images of calcein, tetramethylrhodamine methyl ester (TMRM), and propidium iodide (PI, arrows) were simultaneously collected. Polarized mitochondria take up red fluorescing TMRM while simultaneously excluding green fluorescing calcein due to the closed conformation of permeability transition pores. After 4 hours of ischemia, anoxia depolarized the mitochondria and TMRM fluorescence was undetectable. At the same time, the mitochondria in the green channel appeared as dark and round voids where each void represents a single, polarized mitochondrion, indicative of the absence of MPT onset during ischemia. After reperfusion, the mitochondria transiently repolarized within 10 minutes, but the MPT initiated thereafter, as shown by the loss of TMRM fluorescence and diffusion of cytosolic calcein into the mitochondria. Both calcein and TMRM fluorescence completely vanished at 50 minutes and PI labeled the nuclei (arrows) due to the loss of the plasma membrane integrity.