Expression of C/EBPα in DC and DC progenitors. (A) Relative quantitative RT-PCR of RNA from sorted KSL (cKit^Hi, Sca1⁺, Lin^–), CMP (Lin^–, Sca1^–, cKit^Hi, CD34⁺, FcγRII/III^–/lo), GMP (Lin^–, Sca1^–, cKit^Hi, CD34⁺, FcγRII/III^hi), CDP (Lin^–, cKit^lo, Flt3⁺, CD115⁺, CD11c^–), preDC (Lin^–, B220^–/lo, CD11c⁺, MHCII^–, CD11b/Mac1^–), cDC (CD19^–, CD3^–, NK^–, B220^–, CD11c⁺, MHCII⁺), pDC (CD19^–, CD3^–, NK^–, B220⁺, CD11c^lo/+, PDCA-1⁺), B (CD19⁺, B220⁺ splenic B cells), and monocytes (CD19^–, CD11b/Mac1⁺, Gr-1⁺ peritoneal monocytes) to assess expression of C/EBPα. Values are presented relative to that of β2-microglobulin RNA. Data are averages of 4 independent experiments (error bars represent SD). (B) DC loss after deletion of C/EBPα. 10⁶ lysed, WBM cells from poly(IC)-injected MxCre^– C/EBPαfl/fl or MxCre+ C/EBPαfl/fl mice were cultured in Flt3L-supplemented media. After 8 days, viable cells were assessed for relative expression of CD11c and Gr1 using flow cytometry. These representative plots show the impact of C/EBPα deletion on the presence of DC (CD11c⁺). The Gr-1+ myeloid cells can also be seen in these plots. GM-CSF and IL4 cultures with MxCre⁺ C/EBPαfl/fl WBM cells resulted in no viable cells after 9 days of culture (FACS plot not shown). (C) Shown are the numbers of total live CD11c⁺ DCs present in the FL or GM-CSF/IL4 cultures for pDC (black, B220⁺) and cDC (white) from (B). The numbers were generated by hemocytometer counts combined with flow cytometry analysis. These numbers of DCs represent mean ± SD from these 4 experiments where MxCre^– and MxCre⁺ correspond to MxCre^– C/EBPαfl/fl and MxCre⁺ C/EBPαfl/fl cells, respectively. (D) Flow cytometric analysis showing CD45.2⁺ Mx1-Cre⁺ C/EBPαfl/fl or C/EBPαfl/fl. WBM cells were cultured together with equal numbers of CD45.1⁺ BM cells and Flt3L (200 ng/mL) for 8 days. Similarly, cultured cells were stained for cell surface markers CD45.1, CD45.2, CD11c, and MHCII followed by flow cytometry and cell counts. DCs derived from WBM were identified as CD45.2⁺ CD11c⁺ Gr1^–. The data shown are representative dot plots from 4 experiments with similar results.