NK cell cloning frequency and absolute NK cell proliferation in AFT024 cultures is dependent on the addition of exogenous cytokines. CD34⁺/Lin⁻/DR⁻ cells were plated in limiting dilutions (replicates of 1,200 cells/well, 400 cells/well, 130 cells/well, and 45 cells/well) on AFT024 in 96-well plates with the cytokine combinations indicated. Cultures were maintained with weekly half media changes and fresh cytokines were added weekly except for IL-3, which was only added once at culture initiation. (A) After 35 to 42 days of culture, wells were analyzed using three-color flow cytometry for the presence of CD56⁺ NK cells to calculate the cloning frequency of initially plated CD34⁺/Lin⁻/DR⁻ cells. Cells were gated on viable cells and any well containing greater than 20 absolute CD56⁺ cells was counted as positive. Each bar represents the mean ± SEM cloning frequency from four donors. (B) The absolute number of NK cells per positive well initiated with 130 CD34⁺/Lin⁻/DR⁻ cells is shown for each cytokine combination. Absolute cell counts per harvested well was determined by addition of a known number of polystyrene microspheres to each sample before analysis by flow cytometry as described in Materials and Methods. Each condition represents the mean ± SEM of 20 to 38 individual wells initiated with CD34⁺/Lin⁻/DR⁻ cells derived from four donors. P values listed are for comparisons between adjacent conditions.