Griess Assay for Nitrite Quantification

NO released from cells was converted to nitrite in the culture medium and was determined using Griess reagent [42 (link)]. Cells were cultured in phenol red free DMEM. After treatment with LPS for 16 h, aliquots (50 μL) of culture medium were transferred to 96-well plates and incubated with 50 μL of reagent A [1% (w/v) sulfanilamide (Sigma-Aldrich, St. Louis, MO) in 5% phosphoric acid] for 10 min at room temperature in the dark, and followed by incubation with 50 μL of reagent B [0.1%, w/v, N-1-napthylethylenediamine dihydrochloride (Sigma-Aldrich, St. Louis, MO)] for 10 min at room temperature in the dark. The absorbance of samples was measured at a wavelength of 565 nm using the Synergy4 Plate Reader. Sodium nitrite (0–100 μM), diluted in culture media, was used to prepare the nitrite standard curve.