Nitrite Measurement from Cell Cultures

Our previous studies demonstrated that NO production in glial cells was mainly due to the induction of iNOS [9 (link)]. Therefore, measurement of NO was used to represent the induction process. NO released from cells was converted to nitrite in the culture medium, which was determined using the Griess reagent. In this study, cells were cultured in DMEM without phenol red. After treating cells with cytokines and LPS, aliquots (200 μl) of culture medium were transferred to test tubes and incubated with 100 μl of the reagent A (1% (w/v) sulfanilamide in 5% phosphoric acid, Sigma) for 10 minutes at room temperature in the dark. This was followed by incubation with 100 μl of reagent B (0.1%, w/v, N-1-napthylethylenediamine dihydrochloride, Sigma) for 10 minutes at room temperature in the dark. After mixing, 100 μl of the purple/magenta solution was transferred to a 96-well plate and the absorbance at 543 nm was measured within 30 minutes in a plate reader. The dilution series of sodium nitrite (0-100 μM) was used to generate the nitrite standard reference curve.