AMD3100 blocks CXCL12-elicited chemotaxis in vivo and in vitro. (a) Sixteen mice were immunized with collagen type II (CII) in complete Freund's adjuvant on day 0 and treated with AMD3100 or PBS in a similar way as described in the legend of Fig. 1. In vivo treatment is indicated along the X-axis. On the last day of treatment, a chemotactic assay was performed as described in Materials and methods. On that day, mice were injected with 2 μg of CXCL12 in 1 ml 0.9% NaCl (+) or 0.9% NaCl only (-) in a subcutaneous air pouch. Two hours after chemokine challenge, cells were washed out of the air pouch with 2 ml of PBS/FCS 2% and counted. Counts of the individual mice are shown (circles) and average ± standard error of the mean are indicated for each group (diamonds). (b,c) Dose-dependent inhibition by AMD3100 of CXCL12-elicited chemotaxis on total splenocytes. On day 21 post immunization with CII in complete Freund's adjuvant, spleens of three mice were pooled and a splenocyte suspension was prepared. Cell samples were pre-incubated for 10 minutes with AMD3100 at the indicated concentrations. Then, 5-μm filter inserts were loaded with 106 cells and transferred to a 24-well plate containing 100 ng/ml human CXCL12 in 600μl of buffer per well. After 3.5 h of incubation, the membrane inserts were removed and the cells in the wells were collected and counted by flow cytometry. The numbers of migrated cells of one representative experiment are shown in (b). (c) The experiment was confirmed by three additional experiments and the data of the experiments were pooled and represented as the percentage inhibition ± standard error of the mean of CXCL12-elicited chemotaxis by the indicated concentrations of AMD3100.