Selection of mitochondria based on their light scattering properties and NAO staining, and measurement of membrane potential and ROS production in isolated mitochondria.

<p>(<b>A</b>) In the FSC/SSC plot of the isolated mitochondria, 10000 events were collected within gate R1. (<b>B</b>) To assess the purity of mitochondria, the preparations were analyzed for NAO fluorescence, and almost all of the events in gate R1 were positive for NAO (M2) compared with samples without staining (M1), which confirmed that they were mitochondria. (<b>C</b>) Changes in the membrane potential in isolated mitochondria in the sham group, in the ischemia (I) 2 h, and in the ischemia/reperfusion (I/R) 2 h, I/R 24 h and I/R 72 h groups, as reflected by changes in TMRM fluorescence intensity. (<b>D</b>) Changes in ROS production in isolated mitochondria in the sham group, the ischemia (I) 2 h, and in the I/R 2 h, I/R 24 h and I/R 72 h groups, reflecting the changes in DCF fluorescence intensity. (<b>E and F</b>) The relative changes in TMRM fluorescence intensity and DCF fluorescence intensity. Six animals were included in each group. Each bar represents the means±SD. ^#P<0.05 versus sham; ^⧫P<0.05 versus I 2 h; ^*P<0.05 versus I 2 h, I/R 2 h and sham; ^⋇P<0.05 versus I 2 h, I/R 2 h, I/R 24 h and sham. Control in <b>C</b> and <b>D</b> indicates samples not stained with probes.