5.5. Superoxide Radical (O2●−) Production

Production of O₂^●− was analyzed through the reduction of ferricytochrome C. The remaining volume (~146 mL) of samples was centrifuged at 2000 rpm at 24 °C; the cell pellet was recovered and re-suspended in 5.0 mL of GSe medium (cell homogenate). Then, 250 µL of the cell homogenate was transferred to a 1.75 mL conical microcentrifuge tube (Fisherbrand^TM) and kept on ice. Cells were lysed by vortexing for 2 min. Krebs buffer (0.11 NaCl, 4.7 mM KCl, 12 nM MgSO₄, 12 nM NaH₂PO₄, 25 mM NaHCO₄, and 1 mM glucose) and cytochrome-C (15 µM) were added. Tubes were capped and incubated at 37 ± 1 °C during 15 min in a shaking water bath (Polyscience, Niles, IL, USA). N-ethylmaleimide (3 nM) was added, and the homogenate was shaken to stop the reaction. Tubes were centrifuged at 3000 rpm, 4 °C for 10 min. Supernatant was transferred to polystyrene disposable cuvettes (Fisherbrand^TM), and absorbance was recorded at 560 nm in a spectrophotometer (Beckman Coulter DU 800, Fullerton, CA, USA). A blank without homogenate was included for each sample. Superoxide radical production was calculated, according to the following formula [107]: