Extended self-renewal and synchronous differentiation of primary, fetal liver–derived mouse erythroblasts. (A) Cells from fetal livers of E12.5 mouse embryos were cultivated in serum-free StemPro (Life Technologies) medium plus NutriMix supplement in the presence of stem cell factor (SCF), erythropoietin (Epo; 2 U/mL), and the synthetic glucocorticoid dexamethasone (Dex). Proliferation kinetics of outgrowing erythroblasts were determined by daily measurements of aliquots in an electronic cell counter (CASY) and cumulative cell numbers calculated as described.⁵⁰  (B) Terminal differentiation was induced by replacing proliferation factors with insulin, the glucocorticoid antagonist ZK112993³⁴  plus high levels of Epo (10 U/mL) and Fe₂-Tf (1 mg/mL = 12.5 μM). To monitor morphologic changes in maturing cells, aliquots were withdrawn at daily intervals, cytocentrifuged onto slides, and stained with neutral benzidine (to detect hemoglobin; brownish stain) and histologic dyes.³⁶  Note size decrease and enucleation of mature cells (72 hours, bottom right panel). Photomicrographs were taken using an Axiovert 10 microscope (Zeiss, Oberkochen, Germany) equipped with a 63 × oil-immersion objective lens (numerical aperture 44-07-61; Zeiss). Images are presented at original magnification, × 630. Images were captured with a Sony 3CCD color video camera (Sony, Tokyo, Japan) and prepared for publication with IP Lab Spectrum P software 3.1.1 (Signal Analytics, Vienna, VA). (C) Measurements of the decline in cell volume during differentiation were performed with an electronic multichannel cell analyzer. Appearance of 5-μm peak indicates mature cells with volumes close to that of peripheral blood erythrocytes. (D) Hemoglobin levels during differentiation were quantitated using a photometric assay previously described, and normalized to both cell numbers and cell volume from 50-μL aliquots in triplicate^32,37 ; error bars, SD of mean, n = 4.