Superoxide anion release was quantified using a standard methodology based on cytochrome C reduction [5 (link),32 (link)]. A Tecan spectrophotometer was used to detect the absorbance in culture supernatants at 550 nm after adding 100 μL of cytochrome C (Merck, Milan, Italy) to each well. Comparatively, empty wells were filled with 100 μL of superoxide dismutase (Merck, Milan, Italy) and 100 μL of cytochrome C, and the plate was incubated for 30 min. The O2 rate was quantified as the average standard deviation (%) of nanomoles per decreased cytochrome C per microgram of protein relative to the control (0 line).
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