TruCOUNT analysis of sorted DCs in culture with GM-CSF and IL-3.

Dot plots demonstrate the forward and side scatter profiles of sorted DCs in culture supplemented with GM-CSF and IL-3 (A). Sorted Lin⁻ cells were gated in R1, and TruCOUNT beads in R2 (A). After gating for HLA-DR expression (R3, not shown), Lin⁻HLA-DR⁺ DCs were analyzed for their subset composition according to their expression of CD11c and CD123 (B). Absolute DC counts were calculated as the number of Lin⁻HLA-DR⁺ cells per 10 000 TruCOUNT beads acquired (mean of triplicate, C, left). The absolute counts for DC subsets were calculated in similar fashion, based on the expression of CD11c (C, middle and right). This pattern is representative of 4 separate experiments. For comparison, dot plots of a 4-color immunofluorescent FACS analysis of cultured PBMCs (Lin.FITC, HLA-DR.PE-Cyanin5) demonstrated the persistence of both the CD11c⁺ and, more strikingly, the CD123⁺ DC subsets after 3 days of culture (D). Error bars show SEM. (*, P < .05; **,P < .001).