Screen for inhibitors of TCR-activation-induced CD69 expression. (a) Cells (3.5 × 108) were infected with pTRA-cDNA libraries. Single-cells were cloned after at least four consecutive sortings of the CD69lowCD3+ phenotype. (b) Cells (7.1 × 108) were sorted with high-speed flow sorters (MoFlo) after stimulation and staining with anti-CD69-APC and anti-CD3-PE. The sort gate was set at the equivalent of 1% of satellite control cells that were stimulated but never flow-sorted (shown as R2) to enrich for the CD69lowCD3+ phenotype. After sorting, the desired cells were allowed to rest for 6 days before another round of stimulation and sorting. (c) Cells were split into two populations after the third round of sorting. One half of the cells were grown in the absence of Dox (top left dot-plot) and the other half in the presence of Dox (top right dot-plot). Six days later, CD69 expression was compared following anti-TCR stimulation. The dashed line indicates CD69 level without Dox and the solid line with Dox (bottom graph).