Monocyte-derived dendritic cell culture

For flow cytometry experiments, blood was collected from two healthy donors and one commercial buffy coat donor. For SWATH-MS, blood was obtained from four healthy donors and pooled in various combinations to form three biological replicates. Samples were purified over a ficoll gradient at room temperature to isolate peripheral blood mononuclear cells (PBMCs; Histopaque, Sigma-Aldrich, Poole, UK). PBMCs were plated for 30–60 mins, non-adherent cells removed and the remaining monocytes differentiated into moDCs as described previously using IL-4 and GM-CSF^{14 (link)}. The moDCs were treated with 100 ng/ml of bacterial LPS (Sigma-Aldrich), or left untreated as control. Cells were cultured in sterile RPMI containing 10% fetal calf serum for up to 24 h.