M csf

Primary human pDC and monocytes were isolated from buffy coats of healthy blood donors provided by the Blutbank Springe (Germany) using ficoll density gradient centrifugation and subsequent magnetic activated cell sorting (Diamond Plasmacytoid Dendritic Cell Isolation Kit, CD14⁺ Cell Isolation Kit; Miltenyi Biotec). Following isolation, 2 x 10⁵ pDC were cultivated for 1 h in 200 μl of 10 ng/ml interleukin 3 containing serum-free DC medium (CellGenix) and were then treated as indicated. moDC, GM-CSF MΦ, and M-CSF MΦ were differentiated from 5 x 10⁵/500 μl monocytes for 5 days in serum-free DC medium enriched with 1000 U/ml GM-CSF (granulocyte macrophage-colony stimulating factor, CellGenix) and 1000 U/ml IL-4 (CellGenix), or 80 U/ml GM-CSF, or 100 ng/ml M-CSF (macrophage-colony stimulating factor, Miltenyi Biotec), respectively.

Plasmacytoid dendritic cells (pDC) as well as monocyte-derived cells were isolated and generated from blood samples of healthy donors as described previously [15]. In brief, pDC and CD14⁺ monocytes were isolated using ficoll density gradient centrifugation and magnetic activated cell sorting. CD14⁺ monocytes were differentiated for 5 d in serum-free DC medium (Cellgenix, 20801–0100) in the presence of 80 U/ml GM-CSF (granulocyte macrophage-colony stimulating factor, CellGenix, 1412–050), 100 ng/ml M-CSF (macrophage-colony stimulating factor, Miltenyi Biotec, 130–096-492), or 1000 U/ml GM-CSF and 1000 U/ml IL-4 (CellGenix, 1403–050) to obtain GM-CSF MФ, M-CSF MФ, or moDC, respectively.