Chemotaxis assay and cell surface expression of CD41a. (A) Chemotaxis responses of the CD41a⁺ cell population. CD34⁺ cells were seeded in serum-free liquid culture containing SCF and PEG-rhuMGDF for up to 6 days. The cells were subjected to chemotaxis through 5-μm pores to various concentrations of SDF-1 and stained with an anti-CD41a MoAb (black bars). Checkerboard analysis was performed by adding SDF-1 (500 ng) both in the bottom and in the top well (white bar). This migration is inhibited by preincubating the cells with pertussis toxin (PTX; hatched bar). Data are expressed as chemotaxis index and represent the mean for one representative experiment done in triplicate. (B) Effect of a blocking MoAb against CXCR4 (12G5). The cells were preincubated either with increasing concentrations of 12G5 MoAb (white squares) or an isotype-control MoAb (black squares) before the migration assay. (C) Chemotaxis responses of megakaryocytes. After migation in response to SDF-1 (500 ng/mL), the cells were stained with an anti-CD41a MoAb. The percentage of CD41⁺cells was determined in starting and migrated cells. The results shown are the mean and SD of five experiments.