Role of SFKs, Syk kinase and integrins in MK migration toward a SDF1α gradient. Purified BM-derived mature MKs adherent on fibronectin (20 μg/mL)–coated coverslip were allowed to migrate toward a SDF1α gradient over 3 hours within the Dunn chamber as described in “Cell migration assay.” To investigate the role of SFKs, Syk kinase and integrins, the Dunn chamber outer well were filled with media containing SDF1α (300 ng/mL) and PP1 (10μM), R406 (2μM) or lotrafiban (10μM). (A) Representative differential interference contrast (DIC) images from 6 independent experiments of primary MKs exposed to a SDF1α gradient are shown (scale bar = 20μm). (B) The migration paths over 3 hours were traced. The intersection of the x- and y-axes was taken to be the starting point of each cell path, whereas the source of SDF1α was at the top. (C) The net translocation distance (displacement from the start to the end point) of each cell in the absence or presence of PP1, R406 and lotrafiban is represented. ***P < .005. See also supplemental Figure 1 videos 1 and 2. (D) CXCR4 surface expression of purified BM-derived mature MKs in the absence or presence of PP1, R406, or lotrafiban was analyzed by flow cytometry using a FITC-conjugated anti-CXCR4 antibody. Gray line indicates relevant control antibody; black line, FITC-conjugated anti-CXCR4 antibody. Representative profile and quantification of the percentage of cells expressing CXCR4 are from 3 independent experiments.