In vitro developed NK cells resemble those found in the thymus and not conventional splenic NK cells. Cells from indicated tissues of Rag1^−/− mice, along with in vitro differentiated NK cells were analyzed by cytometry. Gray-filled histograms represent cells stained with an isotype control and black line histograms represent cells stained with the indicated antibody. (A) In vitro generated NK cells were compared with freshly isolated splenic and thymic NK cells. Gated NK1.1⁺ cells are shown. (B) The phenotype of in vitro generated NK cells in culture for 19 days (top panel) was compared with that of cells that were removed from culture after 19 days and rested in LD IL15 for 36 hours (bottom panel). Gated NK1.1⁺ cells are shown. As described for Figure 2 and here, the staining patterns for each marker were somewhat different but we used the percentage of positive cells as a convenient means to compare and describe the staining profiles for numerous antibodies used. Data are representative of 2-5 experiments.