Nitrate reductase enzyme

The formation of nitric oxide, measured as total nitrites, was set as a functional readout and detected in media as total nitrites using Griess reagent as we have described earlier.²⁸ Briefly, the cell culture supernatant (120 µl) was collected after agonists/antagonists treatment and transferred to another clear 96‐well plate and incubated with nitrate reductase enzyme (13 µl) (Cayman Chemical) and cofactor preparation (13 µl) (Cayman Chemical) for 2 hours at 37°C. Samples and nitrite standards (0‐25 µmole/l) (Cayman Chemical) were allowed to react with sulfanilamide (50 µl, 1% in 5% phosphoric acid) (TCI America) for 10 minutes on gentle shaking. The reaction was continued with the addition of N‐(1‐naphthyl)ethylenediamine dihydrochloride (50 µl, 0.1% in distilled water) (Sigma‐Aldrich). Absorbance was immediately read in Varioskan Flash plate reader (Thermo Fisher Scientific) at 540 nm. The total nitrites were normalized to basal and fold change values.