Effect of cell cycle synchronization on BM homing of cultured CD34⁺ cells. (A) Freshly isolated CD34⁺ cells reside in the G₀/G₁ phase of the cell cycle (i). Cells were stimulated with SCF, FL, or TPO for 16 hours, after which they were reversibly blocked at the G₁/S transition by 24-hour treatment with 2 μg/mL aphidicolin (ii) or were kept in initial conditions (iii). Aphidicolin-treated cells were washed extensively and were replated in fresh medium and cytokines to allow cell cycle progression. Cells entered the S phase after 3 hours (iv) and reached G₂/M at 6 hours (v). Cell cycle status was determined by DNA staining with PI, and the percentage of cells in the G₀/G₁ or S/G₂/M phase of 1 representative experiment is shown. (B) Cell populations ii to v, as indicated in panel A, were infused into NOD/SCID (1-2 × 10⁶ cells/animal). Homing of CFCs to the BM was analyzed at 24 hours. Data are given for individual animals in 2 independent experiments as percentage homing of GM-CFCs. Mean values are represented by bars.