IgG dependency of extra-phagosomal superoxide production in human neutrophils. (A) Primed human peripheral blood neutrophils were preincubated with NBT and left untreated (cells alone) or incubated with S aureus opsonized with either 10% normal serum (complete) or serum depleted of IgG (IgG-depleted) before opsonization, as described in the supplemental Methods. Nonphagocytosed S aureus were lysed by lysostaphin, and samples cytospun onto glass coverslips. Samples were prepared and visualized as described in Figure 1. Shown are representative images. Arrows mark extra-phagosomal formazan deposition in response to phagocytosis of S aureus opsonized with complete serum. (Aii) Number of S aureus phagocytosed under each condition (S aureus/cell), and extra-phagosomal formazan deposits (extra-phagosomal formazan/cell) in at least 40 cells from each condition were counted and expressed as mean ± SEM, P < .0001. (B) Human neutrophils were preincubated with luminol/SOD (intracellular) or isoluminol/HRP (extracellular) as described in “Measurement of ROS production,” before addition to S aureus opsonized with either 10% normal serum (complete, comp) or serum depleted of IgG (IgG-depleted, IgG-dep) before opsonization. ROS responses were measured and data are presented as described in Figure 6A. *P ≤ .0001, paired Student t test compared with complete serum-opsonized S aureus–induced responses. (C) Neutrophils were incubated with NBT, left untreated (cells alone) or incubated with IgG-SRBCs, or IgG-opsonized or unopsonized 3-μm beads, as described in the supplemental Methods. Cells were allowed to adhere to coverslips for 3 minutes at 37°C, and samples were prepared and visualized as described in Figure 1. Shown are representative images. Position of the IgG-SRBC phagosome is indicated by a star.