Mitochondrial Function Assessment using Seahorse

Mitochondrial function was studied using the Seahorse XFp analyser (Agilent Technologies, Santa Clara, CA USA). The left kidney was excised, minced and mitochondria were isolated as previously described [17 (link)] in mitochondrial isolation buffer (containing 70 mM sucrose, 210 mM mannitol, 5 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid), 1 mM EGTA (ethylene glycol-bis (betaaminoethylether)-N,N,N’N’-tetraacetic acid) and 0.5% (w/v) fatty acid-free BSA(bovine serum albumin), pH 7.2). The protein content was determined using the Bio-Rad protein assay. Mitochondrial coupling or electron flow experiments were carried out at 37 °C according to Roger et al. [24 (link)] and results were analysed on the Wave 2.3.0 software (Agilent Technologies, Santa Clara, CA USA).