Nitrite Determination in Cell Cultures

NO released from cells was converted to nitrite in the culture medium and determined using the Griess reagent protocol [25] (link). In brief, cells in 96-well plate were serum-starved in phenol red-free DMEM for 3 h, followed by incubation with specified concentrations of SF-E or inhibitors for 1 h, and then treated with IFNγ and/or LPS at 37°C for 16 h. Aliquots of the media (50 µL) were incubated with 50 µL of the reagent A (1% (w/v) sulfanilamide in 5% phosphoric acid, Sigma-Aldrich) for 10 minutes at room temperature covered in dark. This was followed by incubation with 50 µL of reagent B (0.1%, w/v, N-1-napthylethylenediamine dihydrochloride, Sigma-Aldrich) for 10 minutes at room temperature, protected from light, and A₅₄₃ nm was measured using a microplate reader. Serial dilutions of sodium nitrite (0–100 µM) were used to generate the nitrite standard curve.