Figure 3: Detection of Trav11Traj18 transcripts and NKT cells of DN-stage origin in the thymus of E8_III-Cre⁺Rag2^fl/fl mice.

(a) Flow cytometry of viable B220^–CD1d-dimer^–TCRβ^neg–lo DN and DP thymocyte fractions from E8_III-Cre⁺Rag2^fl/fl mice, showing the sorting strategy. Numbers adjacent to outlined areas indicate percent CD5^loCD69^– (pre-selection DP and DN) cells. (b) qPCR analysis of Trav11Traj18 mRNA in DP and DN thymic fractions from E8_III-Cre⁺Rag2^fl/fl mice sorted as in a (results presented as in Fig. 2b). ND, not detected. (c) Flow cytometry of DN thymocytes obtained from E8_III-Cre⁺Rag2^fl/fl mice and purified by depletion of CD4⁺, CD8⁺ and DP cells via magnetic microbeads and assessed by staining with unloaded CD1d dimers (staining control; left) or α-GalCer-loaded CD1d dimers (right). Numbers adjacent to outlined areas indicate percent CD1d-dimer⁺ NKT cells among gated DN TCRβ⁺ thymocytes. (d) Absolute number of CD1d-dimer⁺ DN NKT cells among DN thymocytes from control mice (n = 5) or E8_III-Cre⁺Rag2^fl/fl mice (n = 8). Data are representative of three independent experiments (mean + s.em. of n = 3 biological replicates in b; mean + s.em. in d).