DC and B-lineage cell cloning frequency in AFT024 cultures is dependent on the addition of exogenous cytokines. CD34⁺/Lin⁻/DR⁻ cells were plated in limiting dilutions on AFT024 in 96-well plates with the cytokine combinations indicated for 35 to 42 days. (A) Multiple replicates were analyzed using three-color flow cytometry for the presence of CD1a⁺ 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 CD1a⁺ cells was counted as positive. Each bar represents the mean ± SEM cloning frequency from four donors. (B) Replicates were analyzed for the presence of CD19⁺ cells to calculate the B-lineage cell cloning frequency. Cells were gated on a very low forward and side scatter (Fig 1B designated R2) and any well containing greater than 10 absolute CD19⁺ cells was counted as positive. Each bar represents the mean and SEM cloning frequency from four donors except for the condition using IL-7, FL, and KL (7/F/K) where eight donors were used. All P values shown are compared with the frequency using IL-7, FL, and KL.