Isolation and Differentiation of Human Macrophages

Human monocytes were obtained from 6 healthy volunteers. Human peripheral blood mononuclear cells (PBMC) were isolated from venous blood by density gradient centrifugation on Lympholyte Cell Separation Media (Cedarlane Laboratories Limited, Burlington, ON, Canada). CD14⁺ monocytes were separated from PBMCs by immunomagnetic sorting using anti-CD14 (MACS CD14 Microbeads; Miltenyi Biotec, Auburn, CA, USA) magnetic microbeads [21 (link)]. Immunomagneting sorting efficiency was 98% according to flow cytometry analysis (data not shown). CD14⁺ monocytes were immediately subjected to macrophage differentiation, as described by Tarique and co-workers [22 (link)]. Briefly, CD14⁺ monocytes were cultured in RPMI 1640 medium (Sigma-Aldrich, USA) supplemented with 10% FBS, 1% penicillin/streptomycin (Sigma-Aldrich, USA), 10 mM HEPES (Euroclone, Pero MI, Italy) and 1 mM L-glutamine (Lonza, USA). M1-like Macrophage differentiation was performed with 50 μg/mL GM-CSF (Miltenyi Biotec, Auburn, CA, USA) for 5 days, followed by 4 days of 20 μg/mL LPS + 20 μg/mL IFNγ (Miltenyi Biotec, Auburn, CA, USA) treatment. M0-like macrophages were obtained after 50 μg/mL GM-CSF treatment for 9 days, while M2-like macrophages were differentiated with 50 μg/mL GM-CSF (Miltenyi Biotec, Auburn, CA, USA) for 5 days, followed by 20 μg/mL IL-4 + 20 μg/mL IL-13 (Miltenyi Biotec, Auburn, CA, USA) treatment for 4 days.