Peripheral Blood Mononuclear Cells Isolation (PBMCs), Monocytes Purification, and Mo-DCs Generation

PBMCs were separated by Histopaque-1077® (Sigma-Aldrich, St. Louis, MO, USA) density centrifugation following the manufacturer's instructions. Monocytes were isolated by positive selection with anti-CD14 magnetic beads using the MACS cell separation system (Miltenyi Biotec, Cologne, Germany).

CD14⁺ cells were used after purification (monocytes) or to promote differentiation in dendritic cells by plating them at 10⁶ cells/mL in RPMI 10% iFBS supplemented with 50 ng/mL of IL-4 and GM-CSF (ImmunoTools, Friesoythe, Germany). After 7 days, monocyte-derived dendritic cells (Mo-DCs) were collected and used for lipid antigen presentation assays.