Three-color flow cytometric analysis of IRES-GFP and p56^lckF505-IRES-GFP–transduced CD4 ISP thymocytes cultured in FTOC. CD4 ISP thymocytes were sorted and transduced with the retrovirus expressing either the control IRES-GFP or the p56^lckF505-IRES-GFP construct (both 2.5 × 10⁴ cells input) and cultured in FTOC for 12 days. 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. Comparable percentages of GFP⁺cells were recovered from the FTOC (4.7% IRES-GFP⁺ and 3.2% p56^lckF505-IRES-GFP⁺). The recoveries in absolute numbers were 4.1 × 10⁵ cells from the IRES-GFP and 6.9 × 10⁵ cells from the p56^lckF505-IRES-GFP seeded cultures, respectively. We harvested comparable numbers of GFP⁺ cells (16,400 IRES-GFP⁺ cells and 20,700 p56^lckF505-IRES-GFP⁺ cells, respectively).