Effect of BMS and SS on DC differentiation. (A) Differentiation of DCs on the cultured BMS. BMS was prepared as described in “Materials and methods.” HPCs were placed on top of the BMS monolayer and cultured for 7 days with or without GM-CSF. Cells were collected and analyzed by flow cytometry as described in Figure 1. To eliminate the effect of possible contamination by fibroblasts, only CD45.2⁺ hematopoietic cells were analyzed. Values are the mean ± SE from 3 experiments. (B) Differentiation of HPCs on BMS or SS with GM-CSF. Cells were labeled with APC-conjugated anti-CD11c or anti–Gr-1 antibodies; FITC-conjugated anti-CD45.2 antibody; PE-conjugated anti-IA^b, anti–B7-2, CD11b, and F4/80; and PerCp-conjugated anti-B220 and anti-CD11b antibodies and analyzed on a FACSCalibur flow cytometer. Only CD45.2⁺ cells were evaluated for cell phenotype. Values are the mean ± SE from 3 experiments. (C) Allogeneic MLR. Cells from the cultures described in panel B were isolated using PE-conjugated anti-CD45.2 antibody, anti-PE beads, and 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. Cell proliferation was measured in triplicate by [³H]-thymidine uptake. Values are the mean ± SE. (D) Gr-1⁺ cells were isolated from bone marrow of control C57BL/6 mice using magnetic bead separation technique. More than 92% of these cells were Gr-1⁺CD11b⁺. Fifteen million of the cells were injected intravenously into congenic CD45.1⁺ mice. Spleen and bone marrow were collected 24, 48, and 72 hours after the injection. Each time point included 3 mice. Cells were labeled with antibodies against different surface markers, and the proportions of CD11c⁺IA^b+ DCs, F4/80⁺Gr-1⁻ macrophages, and Gr-1⁺CD11b⁺ immature myeloid cells were evaluated within the population of CD45.2⁺ donor cells. (E) Splenocytes (SP) and bone marrow cells (BM) were collected 24 hours after transfer of Gr-1⁺CD11b⁺ ImCs as described in panel D. Cells were labeled with APC-conjugated anti–Gr-1, PE-conjugated anti-Neu, PerCp-conjugated anti-CD11b, and FITC-conjugated CD45.2 antibodies and analyzed on a FACSCalibur flow cytometer. The proportions of cells were calculated within the population of donor's CD45.2⁺ cells. Before indicates cells analyzed prior to the transfer. Results of 2 performed experiments are shown.