Figure 1
C5aR−/− dendritic cells (DCs) show reduced antigen uptake. (a) Experimental design. Briefly, bone marrow (BM) cells were isolated from wt BALB/c and C5aR−/− mice and differentiated for 10 days in the presence of 20 ng ml−1 granulocyte macrophages colony-stimulating factor (GM-CSF). On day 10, 3 × 105 DCs were incubated for different periods with either fluorescein isothiocyanate (FITC)- or DQ-OVA (ovalbumin) to assess antigen uptake or processing. Cells were washed, stained with anti-CD11c-APC and anti-CD11b-APC-Cy7 and analyzed by flow cytometry. Cells incubated at 4 °C served as controls. As a measure of uptake/processing the ΔMFI (mean fluorescent intensity) was calculated. (b) Determination of antigen uptake using FITC-OVA (left panel) and antigen uptake and processing using DQ-OVA (right panel). (c) Kinetic of antigen processing by wild-type (wt) or C5aR−/− DCs. Histograms are representative of three independent experiments. (d) To differentiate between pinocytosis and scavenger-mediated endocytosis, BM cells were treated with dimethylamiloride (DMA) to block pinocytosis and poly-inosinic acid (polyI) to block scavenger-mediated endocytosis 30 min before incubation with either FITC-OVA (left panel) or DQ-OVA (right panel) for 240 min. Values shown are the mean±s.e.m.; n⩾3 per group, *P<0.05, **P<0.01, ***P<0.001.
