Binding and uptake of hD1-KLH by DCs. (A) iDCs were treated with 10 μg/mL hD1-KLH, 100 μg/mL AZN-D1, or pretreated with 100 μg/mL AZN-D1 followed by 10 μg/mL hD1 incubation, followed by incubation with an Alexa Fluor 647–labeled goat anti–human IgG antibody. Binding of hD1-KLH was analyzed by flow cytometry. (B) Internalization of hD1-KLH was confirmed by CSLM. iDCs were incubated with Alexa Fluor 647–labeled hD1-KLH (blue) for one hour at 37°C, followed by microscopic analysis. The image represents the middle focal plane of the DCs, with iris set at 2 nm. Original magnification, ×600. (C) iDCs were incubated with Alexa Fluor 488–labeled KLH for one hour at 4°C or 37°C. Subsequently, cells were fixed and analyzed by flow cytometric analysis. (D) iDCs were labeled with LysoTracker Red, followed by addition of Alexa Fluor 488–labeled (green) hD1-KLH or KLH. Cells were imaged by CSLM. Data represent bright field and corresponding fluorescent images of cells at various time points after addition of hD1-KLH or KLH to the culture medium. Original magnification, ×630.