GFLC fractionation. (A) Flow cytometric analysis of GFLC-sorted cells. Cells were first gated based on forward and side scatter to eliminate debris. The plots show intensities of c-kit–APC levels on the y-axis and TER-119–PE levels on the x-axis. Pink lines separate the quadrants (eg, c-kit–APC⁺ plus TER-119–PE⁺ on the top right, etc). Lines were positioned so that 95% would be in the bottom left (negative) quadrant in the unstained control. (B) Histograms show the TER-119–PE intensities of GFLC fractions. The plots show cell numbers on the y-axis and TER-119–PE levels on the x-axis. (C) Distribution of cell size in GFLC fractions. Fraction of max (y-axis) was estimated as follows: cell numbers at each cell volume were counted and normalized by peak cell numbers in each fraction. Data smoothing was performed (n = 7). (D) Wright-Giemsa–stained cytospin preparation of GFLC fractions. Fraction 1 consists predominantly of enriched enucleated reticulocytes and late orthochromatophilic erythroblasts. Early orthochromatophilic and polychromatophilic erythroblasts become more prominent in fraction 2. Fractions 3 and 4 are enriched in late polychromatophilic and basophilic erythroblasts. Fractions 5 and 6 are enriched in larger cells containing basophilic erythroblasts. Image acquisition information can be found in Document S1.