Quantification of inducible nitric oxide synthase (iNOS) activity – Griess assay

The activity of iNOS was assessed with the Griess assay²⁴. The Griess assay detects nitrite ions formed as a byproduct of nitric oxide, reflecting the enzyme’s activity (iNOS). The media were incubated with nitrate reductase from the mold Aspergillus Niger (50 mU/mL, Sigma-Aldrich, St. Louis, MO, USA) and NADPH (Sigma-Aldrich, St. Louis, MO, USA, 100 µmol/L) for 30 min at room temperature. After reduction, the samples were incubated with methanol and diethyl ether (a 3:1 mixture, v/v) for 1 h at 4 °C. After incubation, samples were centrifuged (10 000 g, 10 min at 4 °C), and the supernatants were used for nitrite determination. A Griess reagent; a mixture of solution A (0.1% N-1-naphthyl ethylenediamine dihydrochloride in water) and solution B (1% sulfanilamide in 5% H₃PO₄, 1:1, v/v) was added to the prepared samples. Griess assays were conducted in duplicate from each sample. Absorbance was measured at 540 nm using a microplate reader (Multi-DetectionMicroplate Reader, Synergy 2, BioTek, Bad Friedrichshall, Germany). A standard curve was generated with sodium nitrite at 2.5 to 150 µmol/L concentrations.