Detection of NK1.1⁺ TCRαβ^dim thymocytes in ZAP-70^−/−/DO10 TCR transgenic mouse.

(A) Thymocytes obtained from ZAP-70^+/−, ZAP-70^−/−, or ZAP- 70^−/−/DO10 mice were stained with PE–anti-NK1.1/FITC–anti-TCRαβ (i), PE–anti-NK1.1/FITC–anti-CD4 (ii), PE–anti-NK1.1/FITC–anti-CD8 (iii), and PE–anti-CD4/FITC–anti-CD8 (iv), and analyzed. Proportions of NK1.1⁺ TCRαβ⁺, NK1.1⁺TCRαβ⁻ (upper and middle panels), or NK1.1⁺ TCRαβ^dim populations (lower panel) are indicated in the leftmost panels on the top of each squared region. Results are representative of 3 separate experiments. (B) Flow cytometric analysis and RT-PCR detection of transgenic TCR in NK1.1⁺ TCRαβ^dim thymocytes in DO10/ZAP-70^−/− mice. Thymocytes of DO10/ZAP-70^−/− mice were stained with PE–anti-NK1.1 antibody and biotinylated KJ1-26 followed by streptavidin-FITC. Dead cells were electronically gated out with propidium iodide staining. The NK1.1⁺ TCRαβ^dim thymocytes (NKT) and NK1.1⁻ TCRαβ⁺ thymocytes (T) (demarcated with squares in the left panel) were sorted, and the expressions of DO10-specific TCRα chain (VαJα_DO) and Cα were examined with RT-PCR.