Progression of cell–surface marker expression in developing erythroblast cultures. Aliquots of proliferating cells were harvested at the times indicated, stained with combinations of fluorescently labeled antibodies against markers characteristic for different hematopoietic lineages and stages of development, and subjected to flow cytometry. (A) Histograms showing the changes in CD117 (c-Kit) and CD71 (transferrin receptor) expression over time. Light gray histograms, background fluorescence. (B) Quantitation (as percentage of positive cells) of erythroid (left panel) versus selected nonerythroid marker expression (right panel) during prolonged in vitro cultivation (see also supplemental Table S1). To eliminate nonspecific staining of myeloid cells by the glycophorin antibody, the fraction of cells expressing the erythroid-specific protein glycophorin A (GPA⁺) is expressed as percentage of transferrin receptor–positive cells. (C) Quantitation of pluripotent (CD34⁺; gated to be CD45^dim) hematopoietic progenitors over time. The fraction of CD34⁺ cells within the myeloid (CD33⁺; left panel) and lymphoid compartment (CD38⁺; middle panel) is shown. Right panel shows total numbers of CD34⁺ cells for comparison, to depict the overall abundance of immature hematopoietic progenitors during the early phases of erythroblast culture.