2.11. Determination of Nitrite Levels as an Estimation of Produced Nitric Oxide

Nitrite levels (NO) were determined by the Griess method as an estimation of the nitric oxide produced. Briefly, 60 μL aliquots of supernatants from the anti-promastigote or anti-amastigote assay were recovered, and 60 μL of Griess reagent (1% sulfanilamide and 0.1% of naphthyltylamidine amino acid in orthophosphoric hydrochloride (H₃PO₄) 5%) was added. After 10 min of incubation at room temperature, the samples were placed in 96-well microplates. A standard curve was made using serial dilutions of NaNO₂, and the absorbance was determined at 550 nm in a microplate reader (Thermo Scientific, Multiskan GO).