Notch-1^-/- ES cells have reduced capacity to differentiate into DCs. (A) Whole cell lysates were prepared from Notch-1⁺/+ and Notch-1^-/- ES cells, and the presence of Notch-1 protein was determined by Western blotting as described in “Materials and methods.” (B) Embryonic bodies were cultured with GM-CSF and IL-3 for 25 days, followed by a 5-day incubation with TNF-α. Cells were labeled with allophycocyanin (APC)–conjugated anti-CD11c, PE-conjugated anti-CD11b, and fluorescein isothiocyanate (FITC)–conjugated anti-IA^b or anti–B7-2 antibodies. Three experiments with the same results were performed. (C) DCs were generated from ES cells as described, irradiated at 150 Gy, and cultured with lymph node (LN) cells from control allogeneic BALB/c mice at different ratios. Cell proliferation was measured in triplicates as described in “Materials and methods.” Values are the average ± SE from 2 experiments.