Macrophages are not required for erythroid enucleation. (A,B) Native erythroblast islands from disaggregated wild-type (left) or Rb^−/− (right) E12.5 fetal liver were examined 16 hours after culture, using anti-F4/80 to stain macrophages (red) and anti-TER119 (green) to stain erythroblasts, and Hoechst 33342 for DNA, indicating no difference in island formation between wild-type and Rb-null-derived cells. (C,D) High-magnification analysis of the interaction between red cells (benzidine-stained brown cells) and macrophages (large “foamy” cells) is examined in the context of 12-day CFU-GEMM colony differentiation derived from wild-type (C) or Rb-null (D) fetal liver progenitors. We noted that Rb-null erythroblasts (D) failed to enucleate irrespective of macrophage contacts (arrow) and demonstrated increased size and abnormal chromatin structure as reported previously.^11,12  (E,F) Native erythroblast islands cultured in vitro from mice with the indicated Rb genotypes and at early or later stages of embryonic development were enumerated (F). Only definitive islands in which 5 or more erythroblasts were bound were counted as islands. Numbers of erythroblasts per macrophage were also counted (E). Experiments were carried out in triplicate. Error bars are standard deviations. Significant differences were observed for the number of erythroblasts per macrophage in “late” E13.5 wild-type and Rb-null fetal liver after 4 hours (P = .001) or 16 hours (P = .001) in culture (E). Student t test. (G) Quantitation of enucleation by flow cytometric analysis of all erythroid cells (floating and attached) was carried out after 60 hours in culture, either in the presence of macrophages and erythroblast island formation or in suspension culture. The percentage of enucleated Rb-null cells was significantly reduced relative to wild type in suspension culture (P < .009) and in macrophage coculture (P < .003). (H) Purified erythroblast progenitors from E12.5 fetal livers were cultured for 48 hours in adherence with macrophages or in suspension culture, and then harvested for CFU-E assay in methylcellulose. Viable cells (250) were plated in triplicate per sample. Numbers of CFU-Es formed were compared with cultures seeded with erythroblasts recovered after only 90 minutes in culture (day 0). See ″Fluorescence microscopy and island counts″ for image acquisition information.