Fig. 4

Api is internalized more rapidly by pDC than Der p 1 or OVA. PDC were isolated from buffy coats by positive selection. Then, they were cultured for either A 90 min or B 16 h in the presence of IL-3 at 10 ng/ml and 40 µg/ml Alexa Fluor 555-labeled Api, Der p 1 or OVA. After incubation, pDC were stained with CD123 APC and incubated for 15 min at room temperature in the dark. After washing with PBS, supernatants were completely aspirated and pDC resuspended in 200 µl BSA (2%) and incubated for 30 min on ice in the dark. After centrifugation, supernatants were completely aspirated and 100 µl fixation buffer was added. PDC were incubated for 20 min at 4 °C in the dark before washing with 1 ml permeabilization/wash buffer. Supernatants were removed and pDC resuspended in 100 µl permeabilization/wash buffer and incubated for 15 min at 4 °C in the dark. Cells were washed with 2 ml PBS, supernatants aspirated and pDC resuspended in 150 µl PBS. The cell suspension was then placed on dried, Poly-D-Lysine coated coverslips and incubated for 90–120 min at 37 °C. After incubation, PBS was aspirated from the coverslips and coverslips were let dry at room temperature. DAPI for cell nucleus staining was used as described and the dried coverslips were put onto the mounting medium cells facing down. Coverslips were fixed on the slide using clear nail polish and let dry. For confocal microscopy, a Leica TCS SP8 inverse confocal microscope was used, objective HC PL APO CS2 with 63 × magnification