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.

Monocyte isolation techniques significantly impact the phenotype of both isolated monocytes and derived macrophages in vitro

“…Reagents were titrated for optimal performance. Blocking of non‐specific binding was achieved with purified human IgG (Beriglobin) (CSL Behring, Pennsylvania) …”

Circulating extracellular vesicle‐associated CD163 and CD206 in multiple myeloma

“…From whole blood samples, PBMCs were isolated using densitygradient centrifugation on a Histopaque-1077 gradient (Sigma-Aldrich). 32 The isolated cells were washed once with PBS (2% foetal calf serum) (FCS, ThermoFisher Scientific, Bleiswijk, Netherlands) with 1 mmol/L EDTA, then once PBS/2% FCS and stored at −80°C in RPMI-1640 medium (ThermoFisher Scientific) with penicillin/streptomycin (100 U/100 µg pr mL, Thermo Fisher Scientific) containing 20% FCS and 10% DMSO (Sigma-Aldrich, St. Louis). Peripheral blood mononuclear cells were stained for flow cytometry analysis in a mix of 50% stain buffer (PBS/0.5% BSA/0.09% NaN 3 ) and 50% BD Horizon™ Brilliant Stain Buffer (BD Biosciences, Erembodegem, Belgium) in the dark at 4°C for 30 minutes, washed and fixed with 0.9% formaldehyde for 15 minutes.…”

“…108450CLS) for 15 minutes at 4°C in the dark. 32 To compensate for spectral overlap, single-stained antibody…”

MiSet RFC Standards: Defining a Universal Minimum Set of Standards Required for Reproducibility and Rigor in Research Flow Cytometry Experiments

“…As a result, critical methods of instrument standardization and calibration (27)(28)(29)(30)(31), panel and experimental design (32)(33)(34)(35)(36), sample preparation and controls (37)(38)(39), spillover correction (40), and data presentation and publication (41,42) are emphasized in various seminal publications. As a result, critical methods of instrument standardization and calibration (27)(28)(29)(30)(31), panel and experimental design (32)(33)(34)(35)(36), sample preparation and controls (37)(38)(39), spillover correction (40), and data presentation and publication (41,42) are emphasized in various seminal publications.…”

“…Simultaneously, substantial efforts were made to communicate best flow cytometry practices through technical publications on standards of instrumentation, experimental design, and data analysis. As a result, critical methods of instrument standardization and calibration (27)(28)(29)(30)(31), panel and experimental design (32)(33)(34)(35)(36), sample preparation and controls (37)(38)(39), spillover correction (40), and data presentation and publication (41,42) are emphasized in various seminal publications. Moreover, more than 40 OMIPs have been published to date, providing a peer-reviewed collection of optimized multicolor panels that can be used in a variety of flow cytometry experiments (18), therefore addressing one of the major sources of variability (9).…”