FACS sorting of Flt3^low and Flt3^high CD34⁺ cells. Cells were stained using antibodies against CD34, CD45, and Flt3 receptor. (A) Gates were first set on light scattering (R1) and fluorescence (R2) to acquire only CD34⁺ cells with low side light scatter and low to moderate forward light scatter properties (“blast gate”) with the isotype control staining for these cells shown in the rightmost panel. (B) With gates R1 and R2 activated, sorting gates were set for acquistion of CD34⁺ cells negative or dimmest for Flt3 receptor (Flt3^low = R3) and CD34⁺ cells brightest for Flt3 receptor (Flt3^high = R4). (C) Reanalysis of CD34⁺ cells obtained after FACS sorting for Flt3 receptor expression using the gates shown in (B). No gates were activated for this reanalysis. Cells obtained by gating on R3 (Flt3^low) are shown in the left plot, and cells obtained by gating on R4 (Flt3^high) are shown on the right. The cell staining profiles in each FACS sorting experiment performed were similar to those shown here. Gates R3 and R4 were consistently drawn to each contain 30% of cells within the total population defined by gates R1 and R2.