Thalassemic erythroid cells differentiated less than similar immature normal erythroid cells in vitro. (A) FACS analysis of wt, th3/+, and th3/th3 splenic erythroid cells before erythroid cell selection. Wt and th3/+ mice were phlebotomized as described in Document S1. (B) FACS analysis was repeated after selection. Numbers on plots are percentages of total cells in the respective gates. (C) Cytospin analysis at time 0 and (D) after culturing the cells for 48 hours in the presence of Epo. Wt cells are all tolidine positive, with the presence of extruded nuclei (arrowhead), and bright tolidine-positive reticulocytes (arrow). The th3/+ sample is characterized by the presence of hemoglobinized polychromatic-orthochromatic erythroblasts (arrowhead), and some rare proerythroblasts (arrow). In the th3/th3 sample, only proerythroblasts/early basophilic erythroblasts (arrow) were detectable, with no presence of tolidine-positive or enucleated cells. For panels C and D, a Plan Fluor 100×/0.75 numeric aperture oil objective was used, along with the same microscope, camera, and software as in Figure 1. (E) CFSE analysis of the erythroid populations. Erythroid cells cultured in the presence of colcemid plus Epo (purple line) or Epo alone (blue line).