BM NKPs can efficiently generate NK and T cells in vitro. Ten Lin⁻CD122⁺NK1.1⁻DX5⁻ NKPs and 5 Lin⁻KIT⁺SCA-1⁺FLT3^high LMPPs sorted from BM of 8- to 12-week-old mice were cultured on OP9 and OP9DL1 stroma cell lines with cytokines: KL, IL-7 (first week only), FL, IL-2, and IL-15. After 14 and 21 days, cells were harvested and evaluated for NK (TCR-β⁻NK1.1⁺DX5⁺ or CD3⁻NK1.1⁺DX5⁺), B (CD19⁺), T (TCR-β⁺NK1.1⁻CD4⁺/CD8⁺ or NK1.1⁻CD3⁺CD4⁺/CD8⁺), and NK1.1⁺TCR⁺ T cells (TCR-β⁺NK1.1⁺DX5⁺) by FACS. ToPro was used to exclude dead cells. (A) Representative FACS profiles of gating and the reanalysis of sorting the Lin⁻CD122⁺DX5⁻NK1.1⁻ NKPs. After gating on Lin⁻CD3⁻ cells, the CD122⁺DX5⁻ gate was set and finally the CD122⁺NK1.1⁻ gate was selected. (Bottom panels) Reanalysis after sorting; the purity was reproducibly more than 98%. (B) Representative FACS profiles of cells generated from NKPs and LMPPs cultured on OP9 and OP9DL1 stroma. The specific gates are indicated in the text above the plot and by the arrow. (C) Mean (SD) proportion of total positive wells and wells containing NK, B, T, and NK1.1⁺TCR⁺ T cells generated from 10 NKPs cultured on OP9 (left panel) and OP9DL1 (right panel). Data represent mean (SD) values from 6 or 7 independent experiments with at least 16 to 24 wells analyzed in each experiment. (D) Proportion of total positive wells and wells containing NK, B, T, and NK1.1⁺TCR⁺ T cells, generated from 5 LMPPs cultured on OP9 (left panel) and OP9DL1 (right panel). Data are from representative experiment with 16 to 36 wells analyzed.