Three-color flow cytometric analysis of IRES-GFP and p56^lckF505-IRES-GFP–transduced CD34⁺thymocytes cultured in FTOC. CD34⁺ thymocytes were sorted, transduced with the retrovirus expressing either the control IRES-GFP or the p56^lckF505-IRES-GFP construct, and cultured in FTOC for 3 weeks. Cell suspensions were stained with PE- and tricolor (TRC)-conjugated antibodies against different surface antigens. Flow cytometric analysis of the control was performed after gating on the GFP⁺ cells (GFP only). Analysis of the FTOC incubated with the p56^lckF505 transduced cells was done following gating on untransduced cells (no Lck-GFP), p56^lckF505-IRES-GFP⁺ cells (intermediate Lck-GFP), and p56^lckF505-IRES-GFP⁺⁺ cells (high Lck-GFP). Numbers in the dot plots and histograms represent percentages of cells. The input was 3 × 10⁴ cells, and after 3 weeks FTOC we harvested 10⁶ cells from the control IRES-GFP transduced and 5 × 10⁵ cells from the p56^lckF505-IRES-GFP–transduced FTOC. This is an expansion of 33-fold and 17-fold, respectively. The recoveries of transduced cells in absolute numbers were 95,000 IRES-GFP⁺ (9.5% GFP⁺) and 32,500 p56^lckF505-IRES-GFP⁺ (6.3% GFP⁺), respectively.