Figure 4.

Cells stimulated with CpGs for 7 days retain capacity to take up CpG ODNs and to translocate them into TLR9-positive endocytic compartments. (A) MPs were either left non-stimulated (blue contour) or were treated with 2 μM CpGs for 7 days (orange contour) prior to incubation with fluorescent (CpG-Cy5) ODNs for 1 h and flow cytometry analysis. The filled gray contour represents cells incubated without fluorescent CpGs. (B) Non-stimulated cells (NS) and cells pretreated with CpGs for 7 days were incubated with CpG-Cy5 for 1 h prior to fixation, permeabilization, and staining of intracellular TLR9. The endocytosed CpGs were visualized in the far-red channel and are shown in green pseudocolor. The nuclei were stained with SYTOX Green (blue pseudocolor). The colocalization between TLR9 and CpG-positive vesicles (yellow color) is indicated with arrows in the merged images.