Ag storage and release by DCs are enhanced by CXCL13 and transfer of Ag from loaded DCs to B cells in vivo. (A-C) Flow cytometric analysis of CXCR5 staining on immature MoDCs (A), primary purified CD19⁺ blood B cells (B), and Jurkat T cells (bold black lines) compared with isotype control (bold gray lines) and unstained cells (thin line) from the same acquisition. (D) MoDCs were pulsed with with 50 μg/mL of Cy2-F(ab)′₂ for 1 hour and then chased for 4 hours at 37°C in the presence of 200 ng/mL of CXCL13 and autologous B cells. Images of MoDCs obtained from ImageStream technology. (E-G) MoDCs were treated as described in Figure 4B through D, except that they were chased for 4 hours in the presence of 200 ng/mL CXCL13. (E-F) Data are mean ± SEM of 3 independent experiments. (G) Graph is representative of 2 independent experiments. (H) Purified DCs from C57Bl/6 mice were pulsed with 50 μg/mL of QDot655-coupled F(ab)′₂ anti–mouse IgG for 1 hour, washed twice, and fixed or not with 1% PFA for 10 minutes before footpad injections with 2.5 × 10⁶ pulsed DCs. PLNs were collected after 24 hours and frozen. Immunostaining of sections was analyzed by spinning disk confocal microscopy (original magnification ×20). CD19 staining for B cells (blue), CD11c staining for DCs (red), and QDot655-coupled F(ab)′₂ (green) are shown. Bar represents 20 μm. Right panels: Magnified images from panel H. (I) CD11c⁺/QDot655-coupled F(ab)′₂⁺DCs and CD19⁺/QDot655-coupled F(ab)′₂⁺ B cells from the unfixed condition were observed (original magnification ×63). Bar represents 5 μm.