Fig. 6. Extracellular oxidants measured by sentinel particles and NBT. (a) The data shows a typical experiment in which an opsonised zymosan particle (labelled '1') is presented to a neutrophil for phagocytosis together with a nonphagocytosed sentinel particle (labelled '2') and another particle near the mouth of the open phagosome (labelled '3'). The upper row shows the phase contrast images, the middle row, the Ca²⁺ images, pseudocoloured as before and the lower row shows the intensity of DCDHF on the particles in the field. The graph on the right shows the complete data for the Ca²⁺ signal (twin peaked) with the arrow marking the time of phagosome closure, and the intensity for the three particles. As before, the intensity of the particle undergoing phagocytosis increased abruptly at the time of the second phase of the Ca²⁺ signal, whereas the other particles fail to respond. (b) The result from a typical experiment in which completion of phagocytosis was prevented by pretreatment of the neutrophils with cytochalasin B (5 µg/ml). The upper series of images show the contact between the zymosan particle and the cell, and lower series, the corresponding cytosolic Ca²⁺ images; the graph on the right shows the complete data for Ca²⁺ signalling and oxidation of the DCDHF-zymosan. Despite inhibition of internalisation, and no detectable oxidation of the zymosan, Ca²⁺ signalling continued normally. (c) The progressive precipitation of formazan during phagocytosis. NBT (1 mg/ml) was added to the cells before the onset of phagocytosis and cells imaged using bright field with weak phase contrast to permit visualisation of the precipitation of formazan, which is seen as black deposit around the phagosome. The white arrow in the first image indicates the exposed portion of the C3bi-opsonised zymosan particle after contact but before complete engulfment. The black arrows indicate the point of phagosome closure, which occurred at 100 seconds and the subsequent images show that the deposition of formazan continued for at least 230 seconds. This experiment was typical of at least three others.