Intraphagosomal measurement of the magnitude and duration of the oxidative burst

Generation of an oxidative burst within the phagosomes of neutrophils, dendritic cells and macrophages is an essential component of the innate immune system. To examine the kinetics of the oxidative burst in the macrophage phagosome, we developed two new assays using beads coated with oxidation-sensitive fluorochromes.These assays permitted quantification and temporal resolution of the oxidative burst within the phagosome. The macrophage phagosomal oxidative burst is short lived,with oxidation of bead-associated substrates reaching maximal activity within 30 min following phagocytosis.Additionally, the extent and rate of macrophage phagosomal substrate oxidation were subject to immunomodulation by activation with lipopolysaccharide and/or interferon-gamma.