Nbt dye

AMs from BALF were grown in a 96-well plate in serum-containing medium at 37°C for 4 h with nitroblue tetrazolium (NBT) dye (Sigma-Aldrich, St. Louis, MO) (1 μg/ml) added into each well. Cells were incubated for another 1 h or until color developed. NBT was reduced by ROS to the dark blue formation, which was dissolved in dimethyl sulfoxide (DMSO), and its absorbance was determined at 560 nm. Similarly, we measured H₂DCF fluorescence to determine the extent of oxidation in AMs (48 (link)).

This assay was used to determine the production of superoxide anion in phagocytic cells. BALF cells were obtained from WT mice and SHIP^−/−mice, then were grown in a 96-well plate in serum-containing medium at 37°C for 4 h, washed three times with PBS buffer, and subsequently infected with PAO1 for 2 h; and 1 μg/ml of nitroblue tetrazolium (NBT) dye (Sigma) was added to each well. The cells were incubated at 37°C for 1 h or until the color developed. The dye was yellow and gave a blue color formazan product upon reduction by superoxide. The reaction was terminated by adding 100 μl of stop solution (10% DMSO; 10% SDS in 50 mM HEPES buffer). The plate was left at room temperature overnight for complete dissolution of formazan product and read at 560-nm absorbance using a multiscan plate reader to quantify the dye conversion. Triplicates were done for each sample and control. Background was corrected by using blanks containing dye alone (32 (link)).