In vitro NKT-ES cell culture. (A) Fluorescence-activated cell sorting (FACS) profiles of cells generated from NKT-ES cells. Cells generated from NKT-ES cells on OP9/Dll-1 in the presence of IL-7 and Flt3L were analyzed by FACSAria at the indicated time points. On days 11 and 14 of culture, Lin (CD3ϵ, CD4, CD8α, TCRβ, TCRγδ, α-GalCer/CD1d dimer, CD11c, CD19, B220, Gr-1)⁻ CD44⁺ CD25⁻ cells, equivalent to the thymic DN1 population, were further separated based on CD45 and CD11b expression. The CD45⁺ CD11b⁻ cells were then analyzed for expression of c-kit and CD24. (B-C) Generation of NKT cells from NKT-ES cells. Indicated fractions in panel A were sorted and further cultured on OP9/Dll-1 with IL-7 and Flt3L for a total of 20 days (B), or in the switch culture on OP9/control with IL-7 and Flt3L starting from day 14 of the original culture for the following 6 days (C). The α-GalCer/CD1d dimer⁺ TCRβ⁺ fraction generated from OP9/Dll-1 (B) or from switch culture on OP9/control (C) was analyzed by FACS.