Monocyte isolation and culture

Peripheral Blood Mononuclear Cells (PBMC) were prepared by centrifugation on a Ficoll gradient (Lymphoprep, StemCell). Blood CD14+ monocytes were isolated from healthy donors' PBMC by positive selection using magnetic beads (Miltenyi). Monocytes (2 × 10⁶ cells/ml) were cultured for indicated times in RPMI‐Glutamax medium (GIBCO) supplemented with antibiotics (penicillin and streptomycin) and 10% Fetal Calf Serum in the presence or absence of 100 ng/ml M‐CSF (Miltenyi), 5 ng/ml IL‐4 (Miltenyi) and 5 ng/ml TNF‐α (R&D Biotechne). Cytokines were added only at the start of the culture, and medium was not refreshed during the course of the culture. In some experiments, monocytes were cultured in the presence of 1 μg/ml of recombinant B18R (StemCell).