2.10. Succinate dehydrogenase activity

Mitochondrial functionality was assessed by performing an MTT assay to measure succinate dehydrogenase (SDH) activity. Isolated kidney mitochondria were suspended in mitochondrial assay buffer (0.5 mmol/L KH2PO4, 20 mmol/L HEPES, 0.5 mmol/L EGTA, 10 mmol/L NaCl, 140 mmol/L KC, 2 mmol/L MgCl2; supplemented with 1 mg/ml rotenone and 10 mmol/L succinate). To initiate the assay, 100 μL of isolated mitochondria (at a concentration of 100 μg/well) were combined with 25 μL of MTT solution (0.5 mg/ml) in 96-well plates. The mixture was then incubated at 37 °C for 30 min. Following incubation, the formazan crystals formed were dissolved by adding 100 μL of dimethyl sulfoxide (DMSO) to each well. The absorbance of the resulting solution was measured at 570 nm using an ELISA reader [17].