Allostimulatory activity of bone marrow-derived plasmacytoid dendritic cells is independent of indoleamine dioxygenase but regulated by inducible costimulator ligand expression

pDC propagation:

BM cells were cultured for 8 days in complete medium in 200 ng/ml Flt3L（CHO cell derived，was a gift from Amgen (Seattle,
WA）.

1. On day 4：50% of the supernatant was replaced with fresh, cytokine-containing medium.
2. On day 8：
   • the cells were enriched for B220⁺ cells by incubation with anti-mouse B220-coated immunomagnetic beads (Miltenyi Biotec, Auburn, CA), according to the manufacturer’s instructions, for 15 min at 4℃ and then positively selected  by  passage  through  a  paramagnetic  column  (Miltenyi Biotec), yielding a highly enriched (＞90%) CD11c⁺B220⁺ population.
   • In some experiments, DC were then activated by overnight  culture  (18  hours)  in  granulocyte–macrophage  colony-stimulating factor (20 ng/ml) and CpG (2 μg/ml).
3. pDC were collected from 7- or 8-day-old BM cultures and plated at 5 ×10⁵ cells/500 μl CM overnight.