3. Measurement of nitric oxide production in RAW 264.7 macrophages

The accumulation of NO in the culture supernatants was assayed using Griess reagent. In brief, the cells were treated with various concentrations of WEMF or 0.5 ng/mL LPS for 24 hours. The supernatant was then collected and mixed with the same volume of Griess reagent for 10 minutes at room temperature in the dark. The absorbance was measured at 540 nm on a microplate reader, and NO concentrations were calculated by referencing a standard curve generated by known concentrations of sodium nitrite.31 A fresh culture medium was used as the blank in all experiments.