Generation of DC precursors and DCs from murine Lin⁻c-kit⁺ HPCs stimulated with GM-CSF + TNF-α. (A) Murine Lin⁻c-kit⁺ HPCs were cultured in the presence of GM-CSF + SCF + TNF-α for 6 to 7 days and then in the presence of GM-CSF + TNF-α for another 7 days. At the indicated timepoint, independent aliquots of cells were recovered and processed for analyses of CD11b and CD11c expression by using double-color immunostaining with anti–CD11b-FITC and anti–CD11c-PE. The data represent mean value ± SD of percentage of the two subpopulations observed in more than five experiments. *P < .05 significance as compared with CD11b^−/dullCD11c⁺ cells at the indicated timepoint. (B) The cells were routinely sorted from cultures of GM-CSF + SCF + TNF-α–stimulated murine Lin⁻c-kit⁺ HPCs at day 6 into CD11b^−/dullCD11c⁺, CD11b^+hiCD11c⁺, and CD11b⁻CD11c⁻ cell populations using EPICS ELITE cell sorter (middle panel). The sorted cells were cultured again in the presence of GM-CSF + TNF-α for an additional 5 to 8 days and reanalyzed for the expression of CD11b and CD11c by double-color immune staining (right panel). Quads were set up on the isotype-matched control dot plot and the results are representative of more than 15 experiments.