Cell cycle fractionation of cytokine-stimulated CD34⁺ cells and the effect on homing. (A) Cell cycle analysis of MPB CD34⁺ cells by PI (i) and fractionation using Hst and Py Y staining (ii). Bars indicate gates for G₀G₁ (A) and S/G₂ M (C) subpopulations, and percentage of cells in each gate is given. (Ai) DNA histograms of MPB CD34⁺ cells immediately after isolation (left) and 48 hours after culture (right) by PI staining. (Aii) Cultured CD34⁺ cells were simultaneously stained with Hst and Py Y. The flow histogram shows gates indicating G₀/G₁ (R1) and S/G₂/M (R2) populations of MPB CD34⁺ cells (left). Sort windows to collect G₀/G₁ and S/G₂/M cells are indicated as regions R1 and R2, respectively. The purity of sorted G₀/G₁ cells (top right) exceeded 98% and of S/G₂/M cells (bottom right) exceeded 83%. Results from a representative experiment are shown. (B) Short-term BM homing of CD34⁺ cells in G₀/G₁ or S/G₂/M. Sorted G₀/G₁ and S/G₂/M fractions of cultured CD34⁺ cells were infused (0.5-2.0 × 10⁶ cells/animal) into sublethally irradiated NOD/SCID animals, and BM homing of progenitor cells was assessed as for Figure 2. Cultured cells that were unstained (presort) or stained (presort stained) served as controls. Data are from 4 independent experiments. Each data point represents 1 animal, and numbers and bars indicate the means.