Definition of phases II, III, and IV of G0CD34+ cells in two-dimensional tracking experiments using sequential cytokine stimulation. Sorted cells were stained with PKH2 on day 0 and maintained with either FL or SCF for the first 7 days of culture. On day 7, cells were washed and stained with PKH26 (two dot plots on the left). After that, cells were split and cultured with different cytokines as indicated to the right. Cells traversing to the upper left hand corner were identified as phase II cells (see Fig 5). Cells receiving SCF as the secondary stimulus (middle column) were capable of few additional divisions only and, therefore, defined phase III cells. For ease of distinction, phase III cells were colored red. When cultured cells received IL-3 for a secondary stimulus (right two dot plots), few additional divisions beyond phase III were demonstrable. Cells capable of proliferating six or more times (see Fig 5) defined phase IV cells. For ease of distinction, phase IV cells were colored in blue. Dot plots used for this illustration were from the same experiment depicted in Fig 7.
