Endocytosis via DC-SIGN neck domain leads to decreased routing to the lysosomal compartments. (A) Immature DCs were incubated with Alexa488-conjugated anti-CRD or anti-neck antibodies (green) at 4°C, washed, labeled with Lysotracker to stain the lysosomes (red), and shifted at 37°C to trigger endocytosis. Cells were analyzed by time-lapse confocal microscopy. Snapshots were taken at the indicated time points. Images represent one focal plane in the middle of the cell body. Representative cells from multiple experiments are shown. Scale bar represents 10 μm. (B) Immature DCs were incubated with antineck antibody H200 or DCN46 (green) at 4°C, washed, and shifted to 37°C for 2 hours. After fixation and permeabilization, the CD63⁺ lysosomal compartment (red) was labeled, and the samples were analyzed by confocal microscopy. Note that, at the 2-hour time point, only intracellular molecules are visualized because extracellular membrane-bound antibodies were removed by acid-strip treatment of cells before fixation. Scale bars represent 10 μm.