Thymic progenitors can differentiate into NK 1.1⁺ cells in vitro. Sorted DN1 CD122⁻NK1.1⁻ thymocytes from Rag1^−/− mice were cocultured with OP9 cells and cytokines. (A) Wells were visually and microscopically examined for growth on different days. Images were acquired using a Nikon Diaphot 200 microscope (Nikon) with a Hamamatsu digital camera (Hamamatsu Photonic) and processed using MetaVue imaging software (Molecular Devices Corp). Top panels are at 4× magnification, while bottom panels are at 20× magnification. (B) Growth positive wells by visual inspection were pooled, stained, and analyzed for expression of NK1.1. For the negative controls, cells were either left unstained (gray-filled histograms) or stained using the appropriate isotype antibody (black dotted histogram). Cells were gated based on lymphocyte population by scatter parameters. Data are representative of at least 3 experiments. (C) Kinetic analysis of NK1.1 and CD122 expression on lymphocyte population, gated by scatter parameters. At various culture periods, wells were examined for the indicated markers as described in panel B. Data are representative of 3-5 experiments