Isolation and Stimulation of Plasmacytoid Dendritic Cells

Leukopacks were obtained from the Etablissement Français du Sang. Peripheral blood mononuclear cells (PBMCs) were recovered using density gradient centrifugation. The pDCs were isolated using magnetic beads on AutoMacs (Miltenyi Biotech) as previously described (Ka et al., 2014 (link)). Briefly, pDCs were isolated by depletion of non-pDCs that were retained in the column, while unlabeled pDCs with high purity (90%) were collected in the flow-through. Plasmacytoid dendritic cells were then suspended in RPMI 1640, supplemented with 20 mM HEPES, 10% fetal calf serum, 2 mM L-glutamine, 100 U penicillin/ml, 50 μg/ml streptomycin (Life Technologies), and 10 ng/ml recombinant IL-3 (R&D Systems), as described previously (Dental et al., 2012 (link)) and were stimulated with heat-inactivated (100°C for 30 min) C. burnetii organisms (bacterium-to-cell ratio of 50:1), or CpG-A 10 μg/mL (ODN 2216; InvivoGen) for 8 or 24 h.