Proliferative response of normal HPCs to leukemic cells in vivo. (A) CFSE assay. A total of 10⁸ BMNCs (C57BL/6J, CD45.2⁺) labeled with CFSE were coinjected with or without 5 × 10⁶Notch1-induced leukemia cells (CD45.2⁺GFP⁺) into lethally irradiated recipient mice (SJL.B6, CD45.1⁺). BMNCs were harvested 72 hours after the transplantation to assess the number of cell divisions. BMNCs were stained with lineage markers and Sca-1, and CFSE-labeled cells were analyzed in the gate for CD45.1⁺Lin⁻Sca-1⁺. A representative figure of the flow cytometric analysis is shown. Blue peaks on the right represent undivided cells (parent cells); each peak toward the left side, one cell division or generation. The figure shown is from 1 of 4 experiments with similar results. The proliferation index¹²  in the CD45.1⁺Lin⁻Sca-1⁺ population is shown in the graph. *P < .05. (B) BrdU assay. BrdU was injected intraperitoneally 72 hours after transplantation. BM cells were analyzed 2 hours later, and the proportion of BrdU⁺CD45.1⁺Lin⁻Sca-1⁺ cells was analyzed with flow cytometry. A representative plot is shown, and the figure shown is from 1 of 6 experiments with similar results. Values are mean ± SD. **P < .01 (n = 3 mice/group, t test). (C) 5-FU assay. A total of 150 mg/g 5-FU was injected 12 hours before BM was harvested, and then CD45.1⁺GFP⁻ cells were isolated for the CFC assay 72 hours after transplantation. The total CFC colonies were counted at day 14 with microscopy. The data shown are from 1 of 2 independent experiments (n = 3-5 wells). **P < .01.