(A) Fluorescence microscopic appearance of Lag staining in a representative cell generated in the presence of FL plus TGF-β1, GM-CSF, TNFα, and SCF (see the Materials and Methods). (B, C, and D) Microscopic appearance of cells generated from singly seeded CD34⁺ cells. Purified CD34⁺ cells (obtained by immunomagnetic isolation, see the Materials and Methods) were sorted individually into 96-well plates using a FACSVantage and cultured in serum-free medium in the presence of FL, GM-CSF, TNFα, and SCF, with or without TGF-β1 for 10 days (see the Materials and Methods). The typical microscopic appearance of colonies is shown. (C) CFC classified as greater than 50 cells/colony (see also Fig 9) generated in the absence of TGF-β1. (D) DC colonies generated in the presence of TGF-β1 (see also Fig 9). (B) Immunofluorescence microphotography showing Lag expression of individually harvested CFU-DC.