SDF-1-triggered calcium mobilization and actin-polymerization are up-regulated by FTY720. (A) Effect of FTY720 on SDF-1-excited calcium mobilization. Isolated PB CD34⁺ cells were loaded with the fluorescent dye Fluo-3 in the presence or absence of 100 nM FTY720 as indicated. After stimulation with 100 ng/mL SDF-1, the FL-1 fluorescence was analyzed in 5-second acquisition intervals by flow cytometry. Preincubation of cells with FTY720 lead to a transient increase in the amount of intracellular free Ca²⁺, with the maximum effect of FTY720 on SDF-1-induced calcium mobilization being observed 20 seconds after stimulation with the chemokine. ♦: FTY720 plus SDF-1; ▵: SDF-1 alone; □: FTY720 alone; ○: Control (carrier). Data represent the mean ± SEM of 4 independent experiments. (B) Effect of FTY720 on SDF-1-induced formation of filamentous actin. Isolated PB CD34⁺ progenitor cells were preincubated or not for 90 minutes with 100 nM FTY720 and subsequently stimulated with 10 ng/mL SDF-1 or carrier (PBS/1% BSA). Formation of filamentous actin was analyzed flow cytometrically by fixation of cells at the indicated time points following permeabilization and staining with FITC-conjugated phalloidin as described in “Materials and methods.” Preincubation of cells with FTY720 had a sustained stabilizing effect on the cytoskeleton. As observed in the calcium mobilization experiments, the effect of FTY720 was highest at the 20-second time point. Data represent the mean ± SEM of 3 independent experiments. ♦: FTY720 plus SDF-1; ▵: SDF-1 alone; ○: FTY720 plus PBS/BSA.