Jagged-1 and Delta-1 had opposite effects on DC generation. (A) Expression of Notch ligands on cell lines used in this study. Whole-cell lysates were prepared. Jagged-1 and Delta-1 proteins were detected by Western blotting using specific antibodies as described in “Materials and methods.” M indicates 3T3-MSCV control cell line; J, 3T3-Jagged-1 fibroblasts; E, 3T3-EF control cell line; and D, 3T3-Delta-1 fibroblasts. (B) HPCs were cultured on irradiated 3T3 fibroblasts in 24-well plates with GM-CSF (20 ng/mL) for 7 days. A typical example of the analysis of cell phenotype is shown. (C) Phenotype of DCs. Cells were labeled with APC-conjugated anti-CD11c or anti–Gr-1 antibodies; PE-conjugated anti-CD45 antibody; FITC-conjugated anti-IA^d, anti–B7-2, and anti-CD11b; and PerCp-conjugated anti-B220 and anti-CD11b antibodies and analyzed on a FACSCalibur flow cytometer. Only CD45⁺ cells were evaluated. Values are the average ± SE from 7 experiments. (D) Allogeneic MLR. CD45⁺ cells from experiments described in panel C were isolated using PE-conjugated anti-CD45 antibody, anti-PE beads, and magnetic-activated cell sorting (MACS) columns. The isolated cells were irradiated at 25 Gy and cultured with lymph node cells isolated from control allogeneic C57BL/6 mice at different ratios. DCs generated from BALB/c using GM-CSF and IL-4 were used as a control. Cell proliferation was measured in triplicate with [³H]-thymidine uptake. Values are the mean ± SE. (E) HPCs were cultured on different fibroblasts in the presence of 100 ng/mL FL for 10 days and analyzed as described in Figure 2B. Mean ± SE of 3 experiments is shown.