Study of apoptosis in purified CD34⁺ cells.

(A) Scattergram of forward scatter (FSC) versus right-angle side scatter (SSC), to allow gating on CD34⁺ cells by excluding cell debris (R1). (B) Scattergram of anti-CD33 fluorescence versus SSC gated on R1 to allow gating on CD33⁺ (R2) or CD33⁻ (R3) cells within the CD34⁺ (R1) cell fraction. (C) Scattergram of anti-CD33 versus anti-Fas fluorescence gated on R2, showing the Fas⁺ (R4) and Fas⁻(R5) CD34⁺/CD33⁺ cells. A similar scattergram gated on R3 allowed the quantification of Fas⁺ and Fas⁻ CD34⁺/CD33⁻ cells. (D) Scattergram of FSC versus 7-AAD fluorescence gated on R2, showing 7-AAD^bright (late apoptotic), 7-AAD^dim (early apoptotic), and 7-AAD⁻ (live) CD34⁺/CD33⁺ cells. Similar scattergrams gated on R2+R4 or R2+R5 regions allowed the estimation of 7-AAD^bright, 7-AAD^dim, and 7-AAD⁻cells within the CD34⁺/CD33⁺/Fas⁺or CD34⁺/CD33⁺/Fas⁻subpopulations, whereas scattergrams gated on R3+R4 or R3+R5 regions allowed the estimation of 7-AAD^bright, 7-AAD^dim, and 7-AAD⁻ cells within the CD34⁺/CD33⁻/Fas⁺ or CD34⁺/CD33⁻/Fas⁻subpopulations.