Fig. 3

Time and concentration dependency of allergen uptake in pDC. PDC purified from buffy coats by BDCA4-positive selection were cultured in 96-round-well plates at a density of 1–1.5 × 10⁵ cells/well and cultured in AIM-V medium containing 10 ng/ml IL-3 and Alexa Fluor 555 labeled Api, Der p 1 or OVA. A, B PDC were incubated with 20 µg/ml allergen for 1.5 h, 4,5 h, 18 h, 24 h or 48 h. C, D Allergen concentrations were used as indicated and pDC cultured overnight. After incubation, surface staining was performed with FITC-conjugated Lin-1 and PerCP-Cy5.5-conjugated anti-CD123 as described and analyzed by flow cytometry. PDC were gated as Lin-1⁻ /CD123⁺⁺. Allergen uptake was analyzed as percentage of Alexa Fluor 555-positive pDC and as median fluorescence intensity (MFI) in pDC. Allergen uptake by pDC from n = 3–7 (Der p 1) and n = 3 (Api and OVA) independent experiments is shown in A, C as median of percent, and in B, D as median of MFI. Error bars indicate ranges. (At some time points error bars are too small to be visible.) Significant differences were assessed using Tukeys multiple comparisons test with * p < 0.05, ** p < 0.005 and *** p < 0.0005