Expression of the CXCR4 receptor on CD34⁺cell-derived megakaryocytes by flow cytometry. CD34⁺cells were cultured in a serum-free liquid culture system in the presence of TPO and SCF for 12 days. Cultured cells were stained with a PE-conjugated anti-CXCR4 MoAb (12G5) and either an FITC-conjugated anti-CD61 MoAb or an isotype control IgG and analyzed on FACscan. (A) The side scatter (SSC) versus forward scatter (FSC) of the analyzed cells. R1 represents the gating of the cells with relatively smaller forward and side scatter properties (which correspond to megakaryocytes with lower ploidy level and smaller size); R2 represents the gating of the cells with relatively larger forward and side scatter properties (which correspond to megakaryocytes with higher ploidy level and larger size). (B) and (D) show the coexpression of CXCR4 with CD61 shown in the dot plot as analyzed with gating in R1 and R2, respectively. The figures in these two quadrants represent the percentage of the double-positive cells. (C) and (E) show the expression of CXCR4 shown in the histogram plot as analyzed with gating in R1 and R2, respectively. The staining with control antibody is shown as the solid profile and the staining with the anti-CXCR4 antibody is shown as the thin line. The percentage of positive expression is provided in the inset. MFI, mean fluorescence intensity.