title: Elimination of erroneous results in flow cytometry caused by antibody binding to Fc receptors on human monocytes and macrophages.
abstract: Nonspecific binding of monoclonal antibodies (mAbs) to Fc-receptors on leukocytes is an important cause of background fluorescence in flow cytometry, and failing to block such nonspecific binding can lead to erroneous results. A major part of previous studies on blocking reagents for flow cytometry have been done in mice, and published results are not completely in agreement. In humans, Fc-receptors are found on most leukocytes, with highest abundance on monocytes/macrophages. Therefore, in the present study our aim was to thoroughly investigate the efficiency of different commonly used blocking reagents regarding inhibition of nonspecific binding of mouse mAbs to human peripheral blood mononuclear cells (MNCs) and monocyte-derived macrophages (MDMs). Monocytes and MDMs showed strong nonspecific binding of IgG1 and IgG2a isotypes, but not IgG2b. In contrast, B-cells, T-cells, and NK-cells did not substantially bind any of the mouse isotype control antibodies evaluated (IgG1, IgG2a, and IgG2b). Importantly, we show that binding of IgG1 and IgG2a to monocytes and MDMs can be eliminated by blocking, either with a commercial Fc-blocking reagent, with mouse or human serum, or with mouse or human IgG in high concentration. Previously, isotype controls have been widely used in flow cytometry assays. However, we show that such controls may be highly unreliable, and we believe they should not be used as gating controls, or to determine background signal. Based on these results, as well as considerations of price and applicability, our recommendation is not to use isotypes as gating controls in flow cytometry, but instead to use 100 μg/mL of purified human IgG as blocking reagent for elimination of nonspecific binding of mouse mAbs to human MNCs and MDMs. © 2016 International Society for Advancement of Cytometry.
authors.affiliation: Department of Biomedicine, Faculty of Health, Aarhus University, Denmark.|Department of Clinical Biochemistry, Aarhus University Hospital, Denmark.|Department of Hematology, Aarhus University Hospital, Denmark.
authors.forename: Morten N
authors.initials: MN
authors.lastname: Andersen
authors.affiliation: Department of Biomedicine, Faculty of Health, Aarhus University, Denmark.
authors.forename: Sinan N H
authors.initials: SN
authors.lastname: Al-Karradi
authors.affiliation: Department of Biomedicine, Faculty of Health, Aarhus University, Denmark.|Department of Rheumatology, Aarhus University Hospital, Denmark.
authors.forename: Tue W
authors.initials: TW
authors.lastname: Kragstrup
authors.affiliation: Department of Biomedicine, Faculty of Health, Aarhus University, Denmark.
authors.forename: Marianne
authors.initials: M
authors.lastname: Hokland
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D005189:False Positive Reactions; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D009000:Monocytes / Q000276:immunology; D011961:Receptors, Fc / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D011961:Receptors, Fc
keywords: Fc-blocking; flow cytometry; human mononuclear cells; immunofluorescence; isotype controls; macrophages; nonspecific antibody binding