Primary human neutrophils (PMNs) were isolated as described previously (77). Briefly, whole blood was separated over a continuous Histopaque-1119 gradient (Sigma-Aldrich), and the layers containing neutrophils and PBMCs were separated. The neutrophils were further purified over a discontinuous Percoll gradient (GE Healthcare) and cultured in RPMI 1640 (without phenol red) with 10 mM Hepes and 0.2% human serum albumin at 37°C and 5% CO2. From the PBMCs, monocytes were isolated by CD14-positive selection (Miltenyi Biotec). Monocytes were differentiated into macrophages with human recombinant MCSF (macrophage colony-stimulating factor) (5 ng/ml, Miltenyi Biotec) for 7 days. pDCs were isolated with the Plasmacytoid Dendritic Cell Isolation Kit II (Miltenyi Biotec) according to the manufacturer’s instructions. PBMCs, monocytes, and pDCs were cultured in RPMI 1640 (with phenol red) supplemented with 10% fetal calf serum (FCS), l-glutamine (2 mM), and penicillin/steptomycin (100 U/ml) at 37°C and 5% CO2.

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