Analysis of proliferation profile. (A) CD15⁺CD14^- neutrophils were cultured with GM-CSF, TNF-α, IFN-γ, and IL-4 for 11 days, washed, and recultured with M-CSF alone for an additional 7 days. CD15⁺CD14^- neutrophils and the cultured cells were tested for Ki-67 staining and BrdU incorporation. Ki-67⁺ and BrdU⁺ cells were counted using a FACSCalibur flow cytometer, and the numbers were expressed as percentages. HPB-NULL cells were used as positive controls. Numbers of cultured cells are also indicated. Representative data from 5 independent experiments are shown. (B) CD15⁺CD14^- neutrophils were cultured with GM-CSF, TNF-α, IFN-γ, and IL-4 for 11 days, washed, and recultured with M-CSF alone for another 7 days. CD15⁺CD14^- neutrophils and the cultured cells were incubated with CFSE. Cell division patterns were analyzed using a FACSCalibur flow cytometer. CD8⁺ cells that underwent several rounds of the cell cycle in response to CD3/CD28 beads were used as positive controls. Experiments were repeated 5 times with identical results.