![]() A clear separation of populations (a high resolution index or staining index) may be obtained by proper titration of antibodies and by minimizing background fluorescence. In flow cytometry, the ability to differentiate between “positive” and “negative” events is of major importance. © 2016 International Society for Advancement of Cytometry 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. 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. Previously, isotype controls have been widely used in flow cytometry assays. 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. 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). Monocytes and MDMs showed strong nonspecific binding of IgG1 and IgG2a isotypes, but not IgG2b. 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). In humans, Fc-receptors are found on most leukocytes, with highest abundance on monocytes/macrophages. 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. All rights reserved.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. The conclusion from this overview is that despite nominal differences, the vast similarities between human and mouse eosinophils provide important insights as to their roles in health and disease and, in turn, demonstrate the unique utility of mouse-based studies with an expectation of valid extrapolation to the understanding and treatment of patients.Ĭopyright © 2012 American Academy of Allergy, Asthma & Immunology. In particular, our review will provide a summation and an easy-to-use reference guide to important studies demonstrating that although differences exist, more often than not, their consequences are unknown and do not necessarily reflect inherent disparities in eosinophil function but instead species-specific variations. The goal of this review is to summarize this literature and in some cases provide experimental details comparing and contrasting eosinophils and eosinophil effector functions in human subjects versus mice. We suggest that this perspective is parochial and ignores the wealth of available studies and the consensus of the literature that overwhelming similarities (and not differences) exist between human and mouse eosinophils. ![]() Indeed, many books and reviews dogmatically highlight differences, providing a rationale to discount the use of mouse models of human eosinophilic diseases. This canonical presence nonetheless does not preclude disparities between human and mouse eosinophils, their effector functions, or both. As a consequence, the hematopoietic lineages of both species are invariantly maintained, each with identifiable eosinophils. The respective life histories of human subjects and mice are well defined and describe a unique story of evolutionary conservation extending from sequence identity within the genome to the underpinnings of biochemical, cellular, and physiologic pathways.
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