K mes

Oxygen consumption was measured polarographically in water-jacketed respiration chambers maintained at 37 °C (Oxygraph, Hanstech Instruments, King’s Lynn, UK) as previously detailed [56 (link)]. Following daily calibration with Buffer Z [50 mM K-MES (Sigma M0895), 30 mM KCl (Sigma P4504), 10 mM K₂HPO₄ (Fisher, Hampton, NH, USA, P290), 1 mM EGTA (Sigma E4378), 5 mM MgCl₂-6H₂O (Sigma M2670), 0.005 mM Glutamate (Sigma G8415), 0.002 mM Malate (Sigma M6773), and 0.05% BSA (Sigma A6003), pH to 7.1 at 4 °C] and Na₂SO₄, 10 µL of isolated SS or IMF mitochondria was independently resuspended in 965 µL of buffer Z, containing 20 mM creatine (Sigma C0708) warmed to 37 °C within the oxygraph chamber. Mitochondria were allowed to equilibrate before the addition of 10 µL Malate (272.21 mM; Sigma M7397) and 10 µL pyruvate (500 mM; Sigma P5280) followed by the addition of 5 µL of ADP (48.09 mM; MP Biomedicals, Irvine, CA, USA, 150259) to determine state 3 and state 4 respiration. Respiratory control ratio (RCR) was designated as state 3 respiration divided by state 4 respiration. Values were normalized post hoc to protein content by the Bradford method.

Animals were sacrificed at week 15 by CO₂ asphyxiation and spinal cords were rapidly removed. For respirometric analysis, tissues were kept in ice-cold BIOPS buffer (10 mM Ca EGTA, 20 mM imidazole, 20 mM taurine, 50 mM K-MES, 0.5 mM DTT, 6.56 mM MgCl2, 5.77 mM ATP, 15 mM phosphocreatine, all purchased from Sigma-Aldrich, St. Luis, MO, USA) to preserve the mitochondrial functions and integrity [72 (link)]. For Western blot analysis, spinal cords were frozen in dry ice and stored at –80 °C.

Animals were sacrificed by CO₂ asphyxiation at 20 weeks of age and spinal cords were rapidly removed. To preserve the mitochondrial functions and membranes integrity, tissue was kept in ice-cold BIOPS buffer (10 mM Ca EGTA, 20 mM imidazole, 20 mM taurine, 50 mM K-MES, 0.5 mM DTT, 6.56 mM MgCl₂, 5.77 mM ATP, 15 mM phosphocreatine, Sigma-Aldrich) [77 (link)]. Alternatively, spinal cords were frozen in liquid nitrogen and stored at −80 °C for further use.
Fresh spinal cords homogenates were prepared within 1 h by using a Potter Elvehjem tissue homogenizer in mitochondrial respiration buffer Mir06 (Oroboros Instruments, Innsbruck, Austria) and immediately used for respirometric analysis.