Superoxide Anion Production Assay in Phagocytes

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)).