Quantifying Nitric Oxide in Macrophages

The nitrite accumulated in culture medium was measured as an indicator of NO production in macrophages, based on the Griess reaction system [39 (link)]. Briefly, 100 μL of each supernatant was mixed with an equal volume of Griess reagent (1% sulfanilic acid in 5% phosphoric acid; 0.5% α-Naphthylamine in 5N acetic acid). After being incubated at RT for 5 min, the sample absorbance was measured at 540 nm in an iMark plate spectrophotometer (Bio-Rad, Tokyo, Japan). The concentrations of nitrite present in the samples were calculated from a standard curve (0 to 100 µM NaNO₂) in culture medium.