The mechanism of the entry of dye into neutrophils in the nitroblue tetrazolium (NBT) test.

1 . Washed buffy-coat preparations of human blood suspended in different media were exposed to the Nitroblue Tetrazolium (NBT) and the mechanism of entry of the dye into neutrophil polymorphonuclear leucocytes was investigated. 2. Stimulation of neutrophils with endotoxin and the presence of heparin and/or fibrinogen in the suspending medium were necessary for dye reduction. 3. NBT complexed with and precipitated heparin and/or fibrinogen from solution. The percentage of cells reducing the dye and the degree of precipitation of fibrinogen and heparin depended on the concentration of dye; the critical concentration of NBT necessary for each effect was the same. 4. Electron microscopy of stimulated neutrophils revealed the presence of amorphous material, which was probably complexed NBT, outside the cell, in the process of endocytosis and within membrane-bound vacuoles within the cytoplasm. 5. Neither complement nor immunoglobulins were obligatory for dye reduction in this system. 6. It is suggested that NBT only enters neutrophils in quantities visible by light microscopy, after stimulation which produces phagocytosis of a macromolecular complex of the dye and heparin and/or fibrinogen.