Respiratory burst activity
The fresh blood with EDTA powder was processed for measurement of nitroblue tetrazolium (NBT) within 1 h after of collection.
Reactive oxygen radical production by neutrophils during respiratory burst activity was assayed by the reduction of nitroblue tetrazolium to formazan. Briefly, the blood was mixed with 0.2% NBT (Himedia, India) in equal proportion (1:1) and incubated at 25 °C for 30 min. In 25 μL of this mixture, 500 μL of dimethyl formamide (SRL, India) was added to solubilise the reduced formazan product. Then it was centrifuged at 2000 × g for 5 min and the supernatant was taken. The extent of NBT reduced was measured at 540 nm. Dimethyl formamide was used as the blank.