FACS analysis of MPB mononuclear cells. (A) Forward scatter (size) and side scatter (density) of MPB MNCs showing region R1 in which CD34⁺ cells are located. (B) Scattogram of CD34-tri-color (Fl 3) versus side scatter (SSC) showing only CD34⁺ cells in R2 that were also gated in R1. (C and D) CD34-tri-color (Fl 3) and CD38-PE (Fl 2) expression from R2 showing the gating of CD34⁺ cells based on CD38 antigen density level (R3, R4, and R5). Region R3 used to define CD34⁺/CD38^−/low cells matching the biological control (see Materials and Methods). Region R3 include CD34⁺ cells with PE-CD38 fluorescence more by 20% to 25% than the maximum PE fluorescence of irrelevant isotype-control (log 10¹). Region R4 defines CD34⁺/CD38²⁺ and region R5 defines CD34⁺/CD38³⁺ cells. Regions R3, R4, and R5 were stored and constantly used for all samples to analyze the third antigen and for cell sorting experiments. (C) The gating of only CD34 strong positive cells are shown, where greater than 98% of CD38^−/low cells are located in region R3. (D) The gating of CD34 weak positive cells are shown, in which region R3 lack the CD38⁻ cells.