Cell sorting strategy and purity of sorted fractions.

Stained cells were sorted according to CD34 and CD38 expression (A) or DNA/RNA staining (B) by using a Coulter Elite flow cytometer. For cell sorting, immunomagnetic-purified CD34⁺ cells were gated on a first R1 region by selecting lymphomononuclear cells and excluding dead cells (FSC/SSC dot plot). A second region R2 corresponding to CD34⁺ cells was drawn on a second dot plot (CD34/SSC) gated on R1. (A) The CD34⁺CD38⁻fraction was arbitrarily defined according to the CD38 expression level as the 10% of CD34⁺ cells with the lowest CD38 labeling. Two gates corresponding to CD34⁺CD38⁻ and to CD34⁺CD38⁺ fractions were defined (Ai and Aii, respectively). Five percent of cells at the limits between these populations were excluded from the sort. (B) CD34⁺ cells were also sorted according to simultaneous DNA/RNA staining with Hoechst 33342 and pyronin Y. This cell cycle fractionation allowed isolation of viable [G₀] CD34⁺ cells from [G₁+S+G₂/M] cells. Cells residing in G₀ exhibit a low RNA content and appear at the bottom of the Hoechst/pyronin Y dot plot identified by a low pyronin Y and a low Hoechst staining (Bi). Cells in G₁ show a brighter pyronin Y signal associated with a low Hoechst staining and cells in S+G₂/M display both high Hoechst and pyronin Y staining. Two gates were defined corresponding to [G₀] CD34⁺cells and to [G₁+S+G₂/M] CD34⁺ cells (Bi and Bii, respectively). The purity of the CD34⁺CD38⁻, CD34⁺CD38⁺, [G₀] CD34⁺, and [G₁+S+G₂/M] CD34⁺ sorted fractions was more than 99% as established by the flow cytometry analysis following sorting. A representative analysis of each sorted fraction cell purity was shown on histograms of CD38 and pyronin Y fluorescences (Ai, Aii and Bi, Bii).