Isolation and Differentiation of Primary Monocyte-Derived Macrophages

Primary monocyte-derived macrophages (MDMs) were prepared as previously described (17 (link)). Briefly, peripheral blood mononuclear cells (PBMCs) from healthy donors and CF patients were isolated by Ficoll density gradient, and monocytes were then positively sorted using anti-CD14 monoclonal antibodies conjugated to magnetic microbeads (Miltenyi Biotec), according to manufacturer’s instructions. Monocytes were then suspended in complete medium and incubated for a further 5 days in 96-well plates at a concentration of 10⁶ cells/ml in the presence of M-CSF (50 ng/ml, Miltenyi Biotec) to get differentiated macrophages.