Monocyte-Derived Macrophage Polarization

Leukocyte concentrates from freshly withdrawn peripheral blood of healthy adult human donors were provided by the Institute of Transfusion Medicine (University Hospital Jena, Germany). The experimental protocol was approved by the ethical committee of the University Hospital Jena. All methods were performed in accordance with the relevant guidelines and regulations. PBMC were isolated using dextran sedimentation and Ficoll-Histopaque 1077-1 (Sigma-Aldrich, Taufkirchen, Germany) centrifugation. PBMC were seeded in PBS containing 1 mM Ca²⁺ and 0.5 mM Mg²⁺ in cell culture flasks (Greiner Bio-one, Frickenhausen, Germany) for 1.5 h at 37 °C and 5% CO₂ for adherence of monocytes. For differentiation and polarization of monocytes towards M1- and M2-MDM, published criteria [27 (link)] were used. Thus, M1-MDM were generated by incubating monocytes with 20 ng/mL GM-CSF (Peprotech, Hamburg, Germany) for 6 days in RPMI 1640 supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine (Biochrom/Merck, Berlin, Germany), and penicillin-streptomycin (Biochrom/Merck), followed by 100 ng/mL LPS (Sigma-Aldrich) and 20 ng/mL IFN-γ (Peprotech) treatment for another 48 h. M2-MDM were obtained after differentiation of monocytes with 20 ng/mL M-CSF (Peprotech) for 6 days, and then with 20 ng/mL IL-4 (Peprotech) for additional 48 h of polarization.