Monocyte-derived Dendritic Cell Culture

All DC cultures were carried out at 37°C, 5% CO₂, in RPMI-1640 (Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco BRL), 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (all Sigma-Aldrich). The use of Leukocyte Reduction System (LRS) cones from platelet donations from healthy volunteers was approved by the Newcastle and North Tyneside Research Ethics Committee 2. Peripheral blood mononuclear cells (PBMC) were isolated from LRS cones by density gradient centrifugation on Lymphoprep (Axis-Shield Diagnostics). To generate immature monocyte-derived DC (moDC) as previously described (23 (link), 24 ), CD14⁺ monocytes were isolated from PBMC by positive magnetic selection using anti-CD14 magnetic microbeads (Miltenyi Biotec) and cultured at 0.5 × 10⁶ cells/ml in a 24-well plate in the presence of IL-4 and GM-CSF (50 ng/ml each, Immunotools, Friesoythe, Germany) for 6 days. Medium supplemented with cytokines was refreshed at day 3. CD1c⁺ DC were separated from PBMC using an immunomagnetic negative selection kit (EasySep human myeloid DC enrichment kit; StemCell) and were cultured in a 96-well plate at 6.5 × 10⁴ cells/200 μl.