4.4. Measurement of Nitric Oxide Production

RAW 264.7 cells were seeded into 96 well plates at a density of 5 × 10⁵ cells/mL for 24 h. The medium was replaced with fresh medium containing 1 μg/mL LPS and 3–100 μM SNAH in the test group, followed by incubation for 24 h. Nitrite accumulated in the culture supernatant was measured as an indicator of NO production using Griess reagent. The culture supernatant was mixed with equal volumes Griess reagent (1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v) naphthylethylenediamine-HCl) for 10 min. Absorbance at 540 nm was measured in a microplate spectrophotometer (Bio-Tek, Vermont, VT, USA). Fresh culture medium was used as the blank in all experiments. The amount of nitrite in the samples was determined by referring to a sodium nitrite standard curve.