Effect of Notch ligands on the proliferation and maturation of DN3 and DN4 thymocytes. Thymocytes were depleted for CD4⁺ and CD8⁺ cells by magnetic bead separation, and DN3 and DN4 subsets were isolated by sorting for Lin^– CD44^–CD25⁺ (DN3) or CD44^–CD25^– (DN4) cells to more than 99% purity. Cells were cultured for 7 days on OP9-MigR1, OP9-Jagged1, or OP9-Delta1. (A) Cell recovery over time in culture. ○ indicates OP9-MigR1; ▴, OP9-Jag1; and •, OP9-Delta1. (B) Maturation of DN3 (top row) or DN4 (bottom row) thymocytes monitored by expression of CD4 and CD8 surface molecules. Expression of CD4 and CD8 molecules on normal thymocytes is shown as a staining control. (C) Expression of TCRβ and TCRδ in the recovered DN3 population shown in panel B. The total number of γδ T cells recovered per well was averaged from 3 independent experiments.