Flow cytometric analyses of PKH2-stained CD34⁺-enriched cells. PKH2-stained CD34⁺-enriched cells, either noncultivated (d0) or cultivated in the presence of early-acting cytokines for 2, 3, 4, or 5 days (d2, d3, d4, d5), were measured after labeling with anti-CD34 and anti-CD133 antibodies. The size and the granularity of all cells is plotted in the panels of the first column, and the PKH2 and anti-CD133 staining of the cells located in the live gates (shown in the first column) in the panels of the second column. Quadrants are adjusted according to corresponding isotype controls. Upon cultivation, CD34⁺ cell increase in size and slightly up-regulate CD133 on their cell surface (compare d0 with d2 plots). Starting at day 3, the content of CD133⁺ cells and the intensity of the PKH2 staining diminishes over time. Since PKH2 is a plasma membrane intercalating dye, its staining gets diluted with each cell division; the PKH2 intensity therefore reflects the number of cell divisions a given cell has performed during cultivation. The perpendicular lines should help to cluster cells regarding the number of cell divisions they had performed. The amount of cells depicted in all plots is normalized to the cell numbers of day 0; therefore, plots can be compared semiquantitatively. Note the small population with the weaker PKH2 staining follows the same kinetics as the large brightly stained population, demonstrating the reliability of the PKH2 experiment.