Temperature-dependent HCV-LP entry into imDCs. Monocyte-derived imDCs were incubated with HCV-LPs or insect cell control preparation (GUS Ctrl) for 1 hour at 4°C (for HCV-LP binding) or 37°C (for HCV-LP entry). Then, DCs were washed in ice-cold PBS, fixed, and permeabilized. HCV-LPs were detected with chimpanzee anti-E2 mAb (49F3) and Cy3-conjugated anti–human IgG antibody (red fluorescence). For costaining of cytosolic lysosomal protein, cells were incubated with a mouse anti–LAMP-2 mAb and FITC-conjugated anti–mouse IgG antibody (green fluorescence). Nuclear staining was performed using DAPI (4′,6-diamidino-2-phenylindole; blue fluorescence). Comparative analysis of stained imDCs by confocal laser scanning microscopy revealed temperature-dependent HCV-LP internalization.