Quantification of Superoxide Anion Release

The quantification of superoxide anion release was obtained following a standard protocol based on the reduction in cytochrome C [38 (link)], and the absorbance in culture supernatants was measured at 550 nm using the spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland). Specifically, 100 μL of cytochrome C (Merck, Milan, Italy) was added to all the wells, while 100 μL of superoxide dismutase (Merck, Milan, Italy) and 100 μL of cytochrome C were added to empty wells and the plate was then incubated for 30 min. After that, 100 μL was taken from each well and the absorbance was measured with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) at 550 nm. The O₂ rate was expressed as the mean ± SD (%) of nanomoles per reduced cytochrome C per microgram of protein compared to the control (0 line).