Differential migration of FL HPCs and adult BM HPCs to SDF-1α in chemotaxis assays.

Migrated HPCs in the lower chamber of the assay system were quantitated (A and C) by flow cytometry gating on c-kit⁺cells (for FL HPC) or CD34⁺ cells (for BM HPC) or (B) by counting colony-forming units after plating of migrated cells in methylcellulose. (A) Dose-dependent chemotactic response of FL HPCs and BM HPCs to SDF-1α in the lower chamber. The chemotactic index was calculated as the number of migrated cells in chemokine-containing wells divided by the number of migrated cells in wells containing medium alone. The frequency of responsive FL HPCs was less than 0.2% of all input cells, whereas 15% to 18% of BM HPCs migrated toward SDF-1α at optimal chemokine concentration. (B) FL HPCs migrate avidly toward a chemotactic signal in supernatant from MS5 BM stroma cells distinct from SDF-1α. Undiluted MS5 supernatant or control medium was added to the lower chamber. Some experiments were performed in the presence of anti–SDF-1α (50 μg/mL). (C) Chemotactic response of FL HPC to MS5 supernatant is blocked after the treatment of HPCs with PTX (left panel) and is attenuated in the absence of a chemotactic gradient. Filled squares connected by lines represent individual experiments performed in parallel with the same batch of FL HPC. Empty circles represent the mean of 6 (left panel,P < .05) or 4 (right panel, P > .05) experiments. Broken line represents chemotaxis to control medium. Bars represent mean ± SEM.