Estimation of the number of divisions attained by G₀CD34⁺ cells in a two-dimensional tracking experiment. G₀CD34⁺ cells were stained with PKH2 on day 0 and cultured for 14 days in the presence of IL-3. On day 7, cells were harvested and stained with PKH26 and analyzed (A). The dark vertical and horizontal lines in all three dot plots represent the lower limits of the fluorescence intensities of PKH2 and PKH26, respectively, achieved directly after staining. Using the relative PKH2 fluorescence loss (50% loss with every division along the X axis), it was possible to identify the position of these cells after every division. Thus, cells moving into phase II (upper left hand corner of dot plot) had been through one, two, or three divisions by day 7. Panel B, which was generated on day 14, depicts the same cells previously shown in A after they were allowed to proliferate for an additional week. The relative positions of the vertical lines designating one, two, and three divisions are still in place. Notice how dividing cells “track” along a 45° diagonal direction because both green and red dyes are lost proportionally as the cells divide, such that cells proliferating beyond three divisions now appear below the dark horizontal line representing the lower limit of PKH26 fluorescence. Panel C, which is composed of the same dot plot shown in B, illustrates how divisions 4, 5, 6, and 7 achieved between days 8 and 14 can be calculated along the Y axis using loss of PKH26 for measurement. Phase III cells were identified as those having gone through four or five divisions, and phase IV cells were defined as those having divided in excess of six times. Proliferation of cells compromising phases II, III, and IV was defined as depicted in Fig 6.