NK-cell number and flow cytometric analysis of surface-marker and cyCD3ϵ expression on in vitro–generated NK cells. (A) Dot plot analysis of CD56 versus membrane CD3 expression in cells generated from CD34⁺ CB progenitor cells in the presence of Flt-3L, SCF, IL-7, and IL-15 on a coculture with OP9 or OP9-control stromal cells after 3 weeks of culture. Graph shows the kinetics of the CD3⁻CD56 ⁺ NK-cell number starting from 1000 CD34⁺ CB progenitor cells generated on OP9-DL1 (◇; full line) or OP9-control (■); dotted line) at different time points of culture as indicated on the x-axis. Data are shown as the means (± SD) from 3 independent experiments. There is a consistently higher number of NK cells when cultured on OP9-DL1 stromal cells. (B,C) Flow cytometric analysis of the NK cells obtained after 3 weeks (B) or 5 weeks (C) with OP9-control or OP9-DL1 stromal cells. The histograms represent the fluorescence intensity of the cells that were gated on CD56⁺ NK cells; the fluorescence of the isotype control of the cells cultured on OP9DL1 is shown as a thin dotted line; the fluorescence of the indicated antigen on cells cultured on OP9-DL1 or OP9-control stromal cells is shown as a thick line or as a gray-shaded area, respectively. The isotype control of the cells cultured on OP9-control stromal cells is not shown as it did not differ from the isotype control of the cells cocultured on OP9-DL1 stromal cells.