Figure 5. rPF4 enhances phagocytosis of S. aureus bioparticles by macrophages.

(A) pHrodo-labeled S. aureus bioparticles (100 μg/ml) were preincubated with rPF4 (40 μg/ml) for 30 min at 37 °C, and rPF4-coated bioparticles were added to adherent IC-21 macrophages for 30 min at 37 °C. Non-phagocytosed bacteria were removed, and phagocytosis was determined from fluorescent cell images. Representative images of IC-21 macrophages exposed to rPF4-coated S. aureus (upper panel) and uncoated control bioparticles (lower panel) are shown. Inset boxes show enlarged regions. (B) Dose-dependent effect of rPF4 on phagocytosis of S. aureus bioparticles by IC-21 macrophages. Phagocytosis was quantified as the number of fluorescent particles per cell. Values are means ± S.D. from four random fields determined for each condition from three experiments. ***p< 0.001, ns, no significant difference. (C) Effect of rPF4 on phagocytosis of S. aureus by suspended IC-21 macrophages. Cells were incubated with pHrodo-labeled S. aureus bioparticles pretreated with 40 μg/ml rPF4. Phagocytosis was measured by flow cytometry. Shown are representative histograms of three experiments. (D) Quantification of the effect of rPF4 on phagocytosis of S. aureus bioparticles by suspended IC-21 macrophages from flow cytometry results. Values are means ± S.D. from three separate experiments. MFI, mean fluorescence intensity. ***p< 0.001 (E) pHrodo-labeled S. aureus bioparticles were incubated with rPF4 (40 μg/ml) and added to adherent peritoneal resident macrophages for 1 h at 37 °C. Non-phagocytosed bacteria were removed, and images of three random fields were taken. Representative images are shown. (F) Quantification of the effect of rPF4 on phagocytosis of S. aureus bioparticles by resident macrophages from results shown in (E). Phagocytosis was quantified as the number of fluorescent particles per cell. Data shown are means ± S.D. from three experiments with duplicates. ***p< 0.001 (G, H) Effect of rPF4 on phagocytosis of S. aureus by adherent inflammatory macrophages. Phagocytosis was quantified as the number of fluorescent particles per cell. Data shown are means ± S.D. from three experiments with duplicates. ***p< 0.001