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. 2012 Dec 17;209(13):2367–2381. doi: 10.1084/jem.20120325

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© 2012 Nordenfelt et al.

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Figure 8. — Phagocytosis and intracellular survival of S. pyogenes. (A) Gating strategy for the flow cytometry-based method to measure phagocytosis. Human neutrophils were gated based on light scatter properties (1) and then gated for CD18 (2) before analysis of Oregon Green/DyLight647-labeled bacteria (3). The top right quadrant represents neutrophils associated with extracellular bacteria; the bottom right quadrant represents neutrophils associated with only intracellular bacteria; and the bottom left represents cells that are not associated with bacteria. (B) Two representative image series from three independent experiments are shown for wild-type bacteria opsonized with either human plasma or saliva (30 min phagocytosis, MOI 25). Differential interference contrast images (top left) show the localization of the neutrophils; this is represented as a thin white line in the other images. All bacteria (Oregon Green–labeled) are shown in green, and extracellular bacteria are shown in magenta (Cy3-labeled anti-IgG). Bars, 10 µm. (C and D) Wild-type and H- mutant bacteria were analyzed after opsonization with either plasma, saliva, heat-inactivated (56°C) plasma and saliva, or saliva samples supplemented with pooled polyclonal IgG (10,000 µg/ml). The bars in groups of five represent MOI 1, 5, 10, 25, and 50 (30 min phagocytosis) and the inset in C shows the same data as a line graph. (C) Percentage of the neutrophil population that has fully internalized at least one bacterium. (D) Mean fluorescence intensity of intracellular bacteria as a relative measure of the number of internalized bacteria per neutrophil. Data are representative of two independent experiments. (E) Intracellular survival of wild-type and H- S. pyogenes was measured after 1 h of phagocytosis by human neutrophils. The bacteria (MOI 10) were opsonized with plasma, saliva, or saliva supplemented with polyclonal IgG (10,000 µg/ml). The data show survival relative to the starting point. Significant differences were recorded when wild-type bacteria preincubated with saliva were compared with either wild-type bacteria treated with plasma (P = 0.023), or to the three samples of H- bacteria preincubated with plasma, saliva, or saliva supplemented with polyclonal IgG (P = 0.034, 0.033, and 0.027, respectively). Data are from three independent experiments.