2.7. Hippocampal mitochondrial function
2.7.1. Hippocampal mitochondrial isolation
After MWM test was finished, all animals were sacrificed and hippocampal tissues were removed. These tissues were minced and homogenized with glass handheld homogenizer. Mitochondria were obtained from the rat's hippocampus by differential centrifugation [38]. In the first centrifugation (at 1500 g, 10 min at 4°C), all of the broken cell debris and nuclei were sedimented. Then the supernatant was centrifuged again at 10,000 × g for 10 min. The above layer was discarded. Then mitochondrial pellet was washed and suspended in the isolation medium and centrifuged at 10,000 g for 10 min. Final mitochondrial suspensions were prepared in Tris buffer containing (0.05 …show more content…
Louis, MO), we could determine both mitochondrial outer membrane integrity and cytochrome c oxidase activity. In the colorimetric assay, cytochrome c oxidase via oxidation of ferrocytochrome c to ferricytochrome c could reduce the absorbance at 550 nm. All of the procedures were done according to the manufacturer's method; 20 μg of mitochondrial fraction were applied for each reaction, and for each trial duplicate reactions were done. In order to evaluate total mitochondrial cytochrome-c oxidase activity, the isolated mitochondrial fraction was diluted in the enzyme dilution buffer (10 mM Tris–HCl, pH=7.0, containing 250 mM sucrose) with n-dodecyl β-D-maltoside (1 mM), and then put it on ice for 30 min. The reaction was started by adding ferrocytochrome-c substrate solution (0.22 mM) to the mitochondrial sample. Oxidation of ferrocytochrome-c by cytochrome-c oxidase at 550 nm caused a reduction in absorbance. Activity of Cytochrome-c oxidase were estimated and normalized for the amount of protein per reaction and the results were displayed as units per milligram mitochondrial protein. Mitochondrial outer membrane damage was determined through the ratio between cytochrome-c oxidase activity in the presence and absence of the detergent (n-dodecyl
2.7.1. Hippocampal mitochondrial isolation
After MWM test was finished, all animals were sacrificed and hippocampal tissues were removed. These tissues were minced and homogenized with glass handheld homogenizer. Mitochondria were obtained from the rat's hippocampus by differential centrifugation [38]. In the first centrifugation (at 1500 g, 10 min at 4°C), all of the broken cell debris and nuclei were sedimented. Then the supernatant was centrifuged again at 10,000 × g for 10 min. The above layer was discarded. Then mitochondrial pellet was washed and suspended in the isolation medium and centrifuged at 10,000 g for 10 min. Final mitochondrial suspensions were prepared in Tris buffer containing (0.05 …show more content…
Louis, MO), we could determine both mitochondrial outer membrane integrity and cytochrome c oxidase activity. In the colorimetric assay, cytochrome c oxidase via oxidation of ferrocytochrome c to ferricytochrome c could reduce the absorbance at 550 nm. All of the procedures were done according to the manufacturer's method; 20 μg of mitochondrial fraction were applied for each reaction, and for each trial duplicate reactions were done. In order to evaluate total mitochondrial cytochrome-c oxidase activity, the isolated mitochondrial fraction was diluted in the enzyme dilution buffer (10 mM Tris–HCl, pH=7.0, containing 250 mM sucrose) with n-dodecyl β-D-maltoside (1 mM), and then put it on ice for 30 min. The reaction was started by adding ferrocytochrome-c substrate solution (0.22 mM) to the mitochondrial sample. Oxidation of ferrocytochrome-c by cytochrome-c oxidase at 550 nm caused a reduction in absorbance. Activity of Cytochrome-c oxidase were estimated and normalized for the amount of protein per reaction and the results were displayed as units per milligram mitochondrial protein. Mitochondrial outer membrane damage was determined through the ratio between cytochrome-c oxidase activity in the presence and absence of the detergent (n-dodecyl