2.5. Measurement of NO production
NO production was measured as a nitrite concentration in cell free culture supernatant using a colorimetric assay. Briefly, following treatment with sample and cytokines (IL-1β and IFN-γ) for 48 h, aliquots (100 μL) of culture supernatants were incubated with 100 μL of 1:1 mixture of 1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-naphthyl) ethyldiamine dihydrochloride at room temperature for 5 min, then absorbance at 540 nm was measured using a spectrophotometer (Immuno Mini NJ-2300, Japan). The concentration of NO was calculated by a linear standard curve generated from serial dilutions of sodium nitrite in working medium.