4.3.2. Superoxide Concentration Measurement

Superoxide levels were determined by electron paramagnetic resonance (EPR), which allows direct detection of unpaired electrons. In this technique, a spin trapping reagent leads to the formation of a more stable free radical that can be examined by EPR [74]. First, mitochondria isolation was performed as described above. Mitochondrial protein concentrations were measured using the Folin–Lowry method and further allowed normalisation of EPR results. EPR Krebs HEPES buffer was prepared with 25 µM deferoxime (DF) and 5 µM diethyldithiocarbamic acid (DETC). Spin probe Krebs HEPES buffer was prepared with 1 mM 1-Hyroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) to allow detection of superoxide ions. The samples were prepared as follows: the 100 µg of isolated mitochondria were used for two sets of samples with and without 10 µM N-acetylcysteine (NAC, 10 µL) to confirm ROS specific sensitivity. A total of 50 µL of spin probe Krebs HEPES buffer was added to each sample, as well as EPR Krebs HEPES buffer q.s. 1 mL. Samples were incubated for 10 min at 37 °C, snap frozen in a 1 mL syringe in liquid nitrogen, and stored at −80 °C until measurements. Measurements were done with an E-scan (Bruker, Germany) coupled with a temperature controller (Noxygen, Germany) set at 37 °C to mimic in vivo conditions. Comparison of ROS concentrations with and without NAC allowed confirmation of the specific detection of superoxide ions. Values were normalised using protein concentrations and as a percentage of the control. All reagents were purchased from Sigma-Aldrich, Saint-Quentin-Fallavier, France.