Isolation and Characterization of Monocyte-Derived Macrophages

Human peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Ficoll-Paque™ Plus; GE Healthcare) of buffy coat preparations from anonymous donors (Institute of Transfusion Medicine, Ulm University). Monocytes were isolated from PBMCs by adherence on tissue culture treated plastic flasks (Falcon, Corning, NY, United States). Monocyte-derived macrophages were generated by incubation with granulocyte-macrophage colony-stimulating factor (10 ng/ml; Miltenyi Biotec, Bergisch Gladbach, Germany) in Macrophage-Serum Free Medium (M-SFM; Gibco, ThermoFisher Scientific, Schwerte, Germany) for 6 days. Phenotypic characterization by flow cytometry demonstrated that macrophages expressed CD68 (anti–CD68-FITC, clone Y1/82A; BD Biosciences, Franklin Lakes, NJ, United States) and MHCII (anti–HLA-DR-PerCP, clone L243; BD Biosciences) as described (Bruns et al., 2012 (link)).

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Noschka R., Gerbl F., Löffler F., Kubis J., Rodríguez A.A., Mayer D., Grieshober M., Holch A., Raasholm M., Forssmann W.G., Spellerberg B., Wiese S., Weidinger G., Ständker L, & Stenger S. (2021). Unbiased Identification of Angiogenin as an Endogenous Antimicrobial Protein With Activity Against Virulent Mycobacterium tuberculosis. Frontiers in Microbiology, 11, 618278.

Publication 2020

Ficoll-Paque™ Plus; granulocyte-macrophage colony-stimulating factor anti–HLA-DR-PerCP, clone L243

Clone Density gradient centrifugation Donors Ficoll Fitc Flow cytometry Granulocyte macrophage colony stimulating factor Hla dr Human Macrophages Monocytes Peripheral blood mononuclear cells Phenotypic Serum Tissue Transfusion

Corresponding Organization : University Hospital Ulm

Other organizations : Universität Ulm, Pharis Biotec (Germany)

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Variable analysis

independent variables

Density gradient centrifugation (Ficoll-Paque™ Plus) of buffy coat preparations from anonymous donors
Adherence of monocytes on tissue culture treated plastic flasks
Incubation of monocyte-derived macrophages with granulocyte-macrophage colony-stimulating factor (10 ng/ml) in Macrophage-Serum Free Medium (M-SFM) for 6 days

dependent variables

Phenotypic characterization of macrophages by flow cytometry, including expression of CD68 and MHCII

control variables

Macrophage-Serum Free Medium (M-SFM)

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