Accumulation of immature myeloid cells after activation of Notch signaling in HPCs. (A) HPCs were cultured on fibroblasts in 24-well plate with FL and supernatants from control splenocytes for 10 days as described in “Materials and methods.” Cells were then labeled with APC-conjugated anti-CD11c or anti–Gr-1 antibodies, PE-conjugated anti-CD45 antibody, peridinin chlorophyll protein (PerCP)–conjugated anti-B220 antibody, and FITC-conjugated anti-IA^q, anti–B7-2, CD11b, anti-F4/80 or Gr-1 antibodies and analyzed on a FACS-Calibur flow cytometer. Only CD45⁺ cells were evaluated for cell phenotype. Values are the average ± SE from 3 experiments. (B) HPCs were plated on fibroblasts in 96-well plate and incubated for 2 or 4 days with 20 ng/mL GM-CSF Cell proliferation was measured by [³H]-thymidine incorporation. (C-D) HPCs were cultured on fibroblasts in 24-well plate with GM-CSF (C) or M-CSF (D) for 7 or 10 days. In GM-CSF culture (C), 5 ng/mL TNF-α was added 48 hours before cell phenotype analysis. Cells were labeled with APC-conjugated anti-CD11c or anti–Gr-1 antibodies, PE-conjugated anti-CD45, and FITC-conjugated anti-IA^q, anti–B7-2, CD11b, or anti-F4/80 antibodies and analyzed on a FACSCalibur flow cytometer. Only CD45⁺ cells were evaluated for cell phenotype. Values are the average ± SE from 3 experiments. Asterisk indicates statistically significant differences between cells treated with 3T3-MSCV and 3T3-Jagged-1 fibroblasts.