Isolation and Polarization of Human Macrophages

Human monocytes were isolated from de-identified leukopacks obtained from Children’s Hospital Blood Bank (Boston, MA) or the Institute of Transfusion Medicine, University Hospital Jena, Germany, with the use of Ficoll-Histopaque 1077-1 (Sigma-Aldrich, St. Louis, MO). Blood was obtained from healthy human volunteers giving informed consent under protocol #1999-P-001297 approved by the Partners Human Research Committee. The protocols for experiments with macrophages were approved by the ethical commission of the Friedrich-Schiller-University Jena. All methods were performed in accordance with the relevant guidelines and regulations. For differentiation and polarization towards M1 and M2, published criteria were used^{9 (link)}. Briefly, M1 was produced by incubating isolated monocytes with GM-CSF (20 ng/ml) for 6 days in RMPI 1640 (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen, Grand Island, NY), 2 mmol/l l-glutamine (Lonza, Basel, Switzerland), and penicillin–streptomycin (Lonza), followed by LPS (100 ng/ml) plus INF-γ (20 ng/ml) treatment for the indicated times (routinely 48 h). M2 was obtained by incubating monocytes with 20 ng/ml M-CSF for 6 days followed by polarization with 20 ng/ml IL-4 for the indicated times (routinely 48 h).