Evolution 500 spectrophotometer

Superoxide anion production rates were quantified by the initial rate of cytochrome c (Cytc) reduction, as described before [67] (link). The reaction is as follows:
Unless indicated, the components of the cell-free system were added as follows: (2–5 nM Cyt b₅₅₈) membrane fractions, (100–200 nM) trimera and (40 µM) arachidonic acid in 500 µL Phosphate Buffer Saline supplemented with 10 mM MgSO₄ for 4 min of incubation at 25 °C in order to allow the Nox complex assembly. The production was initiated by addition of (250 µM) NADPH and the rate of O₂^−• was quantified by the reduction of cytochrome c (50 µM). The rate was measured at 550 nm in a Thermo evolution500 Spectrophotometer. The amount of superoxide was calculated using a molar extinction coefficient (Δε of the reduced minus oxidized form of Cytc) of 21 mM⁻¹ cm⁻¹. 20 mM stock solution of cholesterol was prepared in ethanol. Further dilutions have been done in ethanol to get a concentration range of cholesterol (0.1–16 mM), which was mixed with AA 65 mM. 3 µL of each mixture was introduced in the cell free system to finally have cholesterol 0.25–40 µM in a mixture with 40 µM AA in the final reaction volume. This allowed keeping the volume constant.