Purification strategy and proliferation of sorted ALL cells in vitro. ALL blast cells were gated on the basis of low forward and side scatter and subsequently gated for expression of CD133-PE and CD19-FITC, R2-R5 (A). Sort gates were separated by at least 10 channels, as shown. Quadrants in dot plot 1 were set up to exclude more than or equal to 99.9% of cells in isotype controls. Reanalysis, after sorting, of CD133⁺/CD19⁻ cells gated in R4 and CD133⁺/CD19⁺ cells gated in R5, is shown in dot plots 2 and 3, respectively. ALL cells from patients 1 to 12 were sorted for expression of CD133 and CD19, and the proliferative capacity of the each of the sorted subfractions and unsorted controls was evaluated in suspension culture (SC) (B). Cultures were maintained with weekly half-media changes. Absolute cell counts, derived from each sorted population and unsorted controls, were determined by flow cytometry at weeks 1, 3, 5, and 6. These values were then used to calculate the proportion of the total viable cells, represented by each sorted population. The proportion of the total cells derived from the each sorted subfraction is presented as the mean plus or minus SE.