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\ the stained samples were mounted on glass slides with a coverslip. The slides were examined by fluorescence microscopy (Olympus America Inc., Center Valley, PA, USA) to confirm the presence of Mitotracker Green inside the mitochondria.
Quantitative analysis of ROS
Cells in the control, 1, 3, and 7 days after ischemic induction were stained with DCF-DA for ROS measurements. Fluorescence was detected using a fluorescent microscope (Olympus America Inc., Center Valley, PA, USA). The amount of ROS was expressed as the fold change from the cells in the control.
Measurement of mitochondrial membrane potential
Mitochondrial membrane potential was detected by loading the cells with JC-1 dye. The distribution and changes in mitochondrial membrane potential were determined by flow cytometry analysis. The cells were examined by flow cytometry in three different groups: 1, 3, and 7 days after the induction of ischemic injury.
Cell viability measurements
The effect of ischemic injury on cell viability was determined by measuring the cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) activity. According to the manufacturer’s directions, the control cells were incubated with MTT solution. The formazan crystals that formed were dissolved in 50 μL dimethyl sulfoxide and added to the wells. The absorbance was measured at 490 nm on a plate reader (Bio-
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