Evaluation of CD34⁺ cell purity by flow cytometry. CD34⁺ cell fractions from peripheral blood (A) and bone marrow (B), separated using MidiMACS columns described in Materials and Methods, were stained with CD45-FITC (J33)/CD34-PE (581) monoclonal antibodies and sequential gating applied. Region A shows CD45 events versus side scatter (SS) that are then analyzed for CD34-PE staining (region B). The CD34⁺ events are then displayed on another CD45 versus SS dot plot (region C), where the CD34⁺ cells form a distinct cluster characterized by low CD45/SS expression. Finally, the events in region C are analyzed by SS and forward scatter (FS) parameters (region D) to define the true CD34⁺ cells. The same gating regions for the CD45-FITC/CD34-PE stained samples were used to analyze the IgG1/FITC isotype control, and these events were subtracted from the number of events in regions A and D to calculate the cell purity.