A. Conserved frequency of Vα14-Jα18 rearrangements in Fyn and SAP-deficient thymocytes. Quantitative RT-PCR of canonical Vα14-Jα18 rearrangements and Cα in sorted DP thymocytes from WT, Fyn-/-, Jα18-/-, CD1-/- (left) and in DP thymocytes from CD1-/-SAP+/+ and CD1-/- SAP-/- mice (right). SAP-/- mice were crossed onto a CD1-/- background to eliminate any contamination of DP thymocyte by mature NKT cells. The values shown are ratios between Vα14-Jα18 and Cα transcripts. B. Vα14-Jα18 transgenic thymocytes on a Fyn+/+, Fyn-/- or SAP-/- background were stained with CD1d-αGalCer tetramers and CD24 to enumerate the immature CD24high and mature CD24low stages. Cell frequencies are indicated in the corresponding gates. C. Left column, CD1d-αGalCer tetramer positive thymocytes were enriched by autoMACS from pools of thymi obtained from 2 week-old SAP+/- and SAP-/- littermates, Fyn-/- and Jα18-/- mice prior to FACS analysis with CD24. Numbers in the CD1d-αGalCer+ CD24high and CD24low gates represent absolute cell numbers recovered from 2 pooled thymi. Right column, gated CD1d-αGalCer+ CD24high cells were analyzed for CD69 expression. Percentages are indicated over corresponding brackets. Similar results were obtained in three independent experiments. D. Stage of NKT cell developmental arrest in the Vα14 Tg SAP-/- thymus. CD1d-αGalCer tetramer+ thymocytes were enriched using paramagnetic beads and submitted to FACS analysis as in 1C.