Figure 5

After binding to B cells, SpA is internalized and induces CXCR4 internalization. (A) B cells were stained with biotinylated Mod-SpA plus AF488-Streptavidin and CXCR4-PE at 4°C for 30 min. One representative staining of three is shown. The percentage of Mod-SpA-positive cells is indicated in the upper right quadrant. (B) B cells were incubated with medium or 100 ng/mL CXCL12 for 30 min at 4°C before staining with CD20-PEcy7 and biotinylated Mod-SpA plus AF488-Streptavidin. Data from one representative experiment of three are shown. The percentage of Mod-SpA-positive cells is indicated in the upper right quadrant. (C) BJAB cells were stained with CXCR4 mAb alone, and then incubated for 2 h with medium alone or 10 µg/mL Mod-SpA at 37°C. Addition of AF488-conjugated goat anti-mouse Ab (green) before or after permeabilization of BJAB cells was used to detect residual surface CXCR4 expression (left panel) or internalized CXCR4 (right panel) by fluorescent microscopy. Nuclei were visualized with DAPI (blue). One representative experiment of two is shown. (D) B cells were stained in medium with AF488-Streptavidin alone or biot-Mod-SpA plus AF488-Streptavidin at 4°C for 30 min then incubated for 2 h at 4°C or 37°C. The residual surface Mod-SpA expression was analyzed by flow cytometry. The percentages and MFI of SpA-positive cells are shown in the upper right quadrant. One representative experiment of three is shown. (E) Location of Mod-SpA (green) was assessed by fluorescent microscopy after incubation at 4°C (left panel) or 37°C (right panel). Nuclei were visualized with DAPI (blue). One representative experiment of three is shown.