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Nitric oxide production was determined by Griess assay to detect in cell culture supernatants. Equal volumes of cell culture supernatant and Griess reagent (0.5 g sulfanilamide (S9251, Sigma-Aldrich), 0.05 g N-(1-Naphthyl)ethylenediamine dihydrochloride (33461, Sigma-Aldrich), and 1.17 mL phosphoric acid per 50 mL water) were mixed in a 96-well plate. Sodium nitrite (237213, Sigma-Aldrich) was used as a standard. Absorbance values were measured immediately at 492 nm using a DTX-880 Multimode plate reader and detection software (Beckman Coulter).
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