Flow cytometric cell sorting.

(A) Flow cytometry identified distinct cell generations following 8-day culture (see Materials and methods for details) of fresh isolated CD34+ cells using CFDA-SE fluorescent dye and Modfit LT software (Verity Software House). Each arrow indicates a distinct cell generation or parental cells and the numbers indicate their frequency in the analyzed cells (expressed as a percentage). (B) Sorting strategy to isolate CLRPP from fresh isolated CD34+ cells cultured for 8 days. CLRPP were sorted on R1 window, setting the sorting windows on the basis of the threshold values of green fluorescence identified previously by the Modfit software for each cell generation. In the typical experiment shown in this figure, CFDA-SE-loaded cultured progenitors were costained with CD34 PerCP to document the maintenance of high levels of CD34 expression on R1 sorted CLRPP. (C) Similarly, costaining with CD105 PE shows the expression of intermediate levels of CD105 antigen on CLRPP and intermediate to high levels of CD105 expression on highly proliferating proerythroblasts (Proery).