pDCs also contaminate the Pre-pro B population.

<p>(A) Flow cytometry analysis was performed on bone marrow cells to subfractionate B220⁺CD19⁻ Fraction A. Using AA4.1, Ly6D and PDCA-1, four populations were distinguished within the B220⁺CD19⁻ population. Each of the gated populations defined were examined for expression of Rag1-GFP mice. The black histogram is the negative control, showing lack of GFP expression in WT mice, with each population color coded to the gating of each population highlighted in A (Purple is B220⁺CD19⁻AA4.1⁻; Red is B220⁺CD19⁻AA4.1⁺Ly6D⁺PDCA-1⁻; Blue is B220⁺CD19⁻AA4.1⁺Ly6D⁺PDCA-1⁺; and Green is B220⁺CD19⁻AA4.1⁺Ly6D⁻PDCA-1⁺). Shown are representative plots of 4 mice from 3 independent experiments. (B) Examination of cell surface markers to distinguish the two populations of B220⁺CD19⁻AA4.1⁺Ly6D⁺ cells. Histograms are color coded as in A and B, with red representing expression in B220⁺CD19⁻AA4.1⁺Ly6D⁺PDCA-1⁻ cells and blue representing expression in B220⁺CD19⁻AA4.1⁺Ly6D⁺PDCA-1⁺ cells. Shown are representative plots from four mice analyzed in two independent experiments. (C) B lineage potential of B220⁺CD19⁻AA4.1⁺Ly6D⁺PDCA-1⁻ cells (red gate in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078408#pone-0078408-g003" target="_blank">figure 3A</a>) and B220⁺CD19⁻AA4.1⁺Ly6D⁺PDCA-1⁺ cells (blue gate in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078408#pone-0078408-g003" target="_blank">figure 3A</a>) was examined by sorting each population onto OP9 stromal cells in media containing IL-7, Stem cell factor (SCF) and Flt3 ligand. After 4 days, cells were stained with CD19 and the total number of CD19⁺ cells generated compared to the number of input sorted cells per well was calculated (frequency of generation of CD19⁺ cells). The frequency was calcuated from 7 independent wells per sample from 3 independent sorts. Statistical analysis was performed using an unpaired two-tailed t test (GraphPad Prism).