Sequential sorting of ex vivo–expanded CD34⁺ cells by cell tracking with PKH2 and cell-cycle fractionation with Hst and PY. Fresh sorted CD34⁺ cells were stained with PKH2 on day 0 (A). Cells were then cultured for 7 days in complete medium supplemented with IL-3-IL-6-SCF (100 ng/mL each). On day 7, harvested cells were stained with CD34-Biotin/streptavidin-APC and analyzed with PKH2 (B). Placement of the vertical cursor was adjusted with reference to day 0 PKH2, and the horizontal cursor with reference to the isotype control. Sorting windows including CD34⁺PKH2^bright and CD34⁺PKH2^dim are indicated. In the second step, sorted CD34⁺PKH2^dim cells were stained with Hst and PY and were further fractionated by DNA/RNA analysis into G₀ , G₁ , and S/G₂ + M phases of the cell cycle (C).