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In-tissue mitochondrial function analysis

Freshly harvested tissues (~10 mg) were immersed in ice-cold BIOPS buffer (10 mM CaK2-EGTA, 7.2 mM K2-EGTA, 20 mM imidazole, 20 mM taurine, 50 mM K-MES, 0.5 mM dithiothreitol, 6.5 mM MgCl2, 5.8 mM ATP, and 15 mM creatine phosphate; pH 7.1). Myofiber bundles were transferred to 2 ml of MIR05 buffer (0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, and 1 mg/ml fatty acid free bovine serum albumin, pH 7.1) containing 50-?g/ml saponin, and incubated at 4°C for 30 min to achieve chemical permeabilization of the sarcolemma membrane. Permeabilized myofiber bundles (~2 mg) were washed with MIR05 buffer, and used for measuring mitochondrial respiration by using an Oxygraph-2k (O2K) respirometer (Oroboros Instruments, Innsbruck Austria). Oxygen concentration was determined at 2 sec intervals and used to compute oxygen flux per mg of tissue by Oroboros DatLab software. Briefly, after recording the baseline respiration with myofiber bundles alone, 5 mM pyruvate, 2 mM malate and 10 mM glutamate (P+G+M) were added, and complex I driven state 4 respiration was recorded. Electron transfer was coupled to phosphorylation by the addition of 5 mM ADP, and state 3 respiration was recorded. Maximal state 3 respiration with parallel electron input from complex I and complex II was recorded with addition of 10 mM succinate, and complex II supported respiration was measured in presence of 6.25 ?M rotenone (inhibits complex I) [22].

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MedpageToday
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02/06/2018