Proliferating cDCs generate CD11c⁻ MHC class II⁻ progeny. Purified BM pre-cDCs were labeled with CFSE and cultured on irradiated stroma derived from neonatal fibroblasts; their progeny were recovered at 3, 6, and 10 days for analysis of CD11c and MHC class II expression (A) and number of cell divisions as determined by CFSE dilution (B). (C) CD11c⁺MHCII⁺ progeny from day 3 cultures in panel A were sorted and seeded on new stroma. Left and right dot-plots show CD11c and MHC class II expression on sorted day 3 cells before culture and their progeny 7 days later. (D) Limiting dilution clonal analysis of purified GFP⁺ pre-cDCs cultured on S17 stromal monolayers for 10 days. Wells with clones containing ≥ 6 cells were deemed positive. Each data point is the mean ± SD of 4 independent experiments. (E) Fluorescence microscopy of clones derived from a single GFP⁺ pre-cDC at 10 days and stained with anti-CD11c or anti-MHC class II. GFP⁺ cDC clones were used as positive controls for CD11c and MHC class II expression. Original magnification, ×20. (F) Pre-cDCs from CD11c-Cre⁺Rosa26-EGFP transgenic mice (white histograms) and their CD11c-Cre⁻ littermate controls (gray histograms) were cultured on stroma. Histograms show CD11c, MHC class II, and GFP expression on the indicated days. Data are representative of 3 independent experiments.