M-CSF drives pDC and cDC development from BM cells, even in the absence of FL. (A) Replicate wells of C57BL/6 BM cells depleted of B220⁺ and CD11c⁺ cells were incubated for 6 days with M-CSF (20 ng/mL) added at day 0 and again at day 3 or with FL (35 ng/mL) added at day 0 only. On each of days 0 to 6, separate wells were stimulated overnight with CpG-2216 or left unstimulated, and the supernatants were assayed for IFN-α. (B) C57BL/6 BM cells depleted of B220⁺ and CD11c⁺ cells were incubated for 6 days with M-CSF (20 ng/mL) added at day 0 and again at day 3. On day 6, the cells were harvested and stained with antibodies to detect CD11c and CD45RA expression. Cells with the phenotype of pDC and cDC populations are shown boxed in the left panel. The number of cells in each of the pDCs and cDCs populations are shown in the right panel and compared with numbers obtained from day 6 FL-generated DC or media only also using BM cells depleted of B220⁺ and CD11c⁺ cells. (C) BM cells from mice lacking FL were similarly depleted of B220⁺ and CD11c⁺ cells and incubated in replicate wells for 0 to 6 days with FL, of M-CSF (with additional feeding at day 3). On each of days 0 to 6, separate wells were stimulated overnight with CpG-2216 or left unstimulated and the supernatants were assayed for IFN-α (left panel). The pDC and cDC populations present at day 6 in the FLKO cultures are shown in right panel. Data shown are from one experiment representative of 3 similar experiments for the multiple timepoints in panel A: one experiment that was carried out for the multiple timepoints and 3 additional experiments for day 6 and 0 timepoints in panel C left: one experiment representative of 3 experiments of day 6 FL cultures and more than 5 experiments of day 6 M-CSF cultures (B), and one experiment that is representative of 4 experiments (C right panel). In media-only control cultures (B,C right panels), no M-DC were detectable.