Analysis of the cell fraction with NKT-cell potential. (A) Reverse-transcription (RT)–PCR analysis. The cells in Fr 5 and Fr 11 shown in Figure 2A were sorted, and total RNA was isolated. The indicated genes were analyzed by RT-PCR using primer pairs listed in supplemental Table 3. Total RNA from 5 × 10², 1 × 10³, and 2 × 10³ cell equivalents was used as template in each lane. Hypoxanthine guanine phosphoribosyl transferase (HPRT) was used as a loading control and the RNA was from 5 × 10² cells. (B) Surface phenotypes. The cells in Fr 11 shown in Figure 2A were analyzed for surface markers by flow cytometry. Shadowed profiles indicated isotype-matched control antibody staining. (C) Intracellular TCRβ staining. The cells in Fr 10 to Fr 14 shown in Figure 2A were analyzed for intracellular TCRβ expression by flow cytometry. Shadowed profiles indicated isotype-matched control antibody staining. (D) FACS analysis of cells derived from NKT-ES-V cells. The developmental progression of NKT-ES-V cells cultured on OP9/Dll-1 under the same conditions as shown in Figure 2A was the same as that of NKT-ES. Thus, cell fractions equivalent to Fr 2, Fr 3, and Fr 5 on day 11 and Fr 7, Fr 8, Fr 10, and Fr 11 on day 14 of culture shown in Figure 2A were gated and analyzed for Venus expression by FACSAria.