Abstract
Monocytes are the third most frequent type of leukocytes in humans, linking innate and adaptive immunity and are critical drivers in many inflammatory diseases. Based on the differential expression of surface antigens, three monocytic subpopulations have been suggested in humans and two in rats with varying inflammatory and phenotype characteristics. Potential intervention strategies that aim to manipulate these cells require an in-depth understanding of monocyte behavior under different conditions. However, monocytes are highly sensitive to their specific activation state and expression of surface markers, which can change during cell isolation and purification. Thus, there is an urgent need for an unbiased functional analysis of activation in monocyte subtypes, which is not affected by the isolation procedure. Here, we present a flow cytometry-based protocol for evaluating subset-specific activation and cytokine expression of circulating blood monocytes both in humans and rats using small whole blood samples (50 - 100 μL). In contrast to previously described monocyte isolation and flow cytometry visualization methods, the presented approach virtually leaves monocyte subsets in a resting state or fixes them in their current state and allows for an unbiased functional endpoint analysis without prior cell isolation. This protocol is a comprehensive tool for studying differential monocyte regulation in the inflammatory and allogeneic immune response in vitro and vivo.
Highlights
Monocytes are white blood cells of the mononuclear phagocyte system, which derive from precursors in the bone marrow and circulate in the blood with a half-life of 1- 2 days before migrating into various tissues to replenish macrophages [1]
To analyze the impact of cell isolation procedures on monocyte activity, we evaluate the conformational change of the CD11b/ CD18 (MAN-1) receptor in monocytes after cell isolation via density gradient centrifugation for isolation of peripheral blood mononuclear cells (PBMC) or fluorescence-activating cell sorting (FACS) for isolation of monocyte subsets
Our study presents a comprehensive flow cytometry-based approach to assess subset-specific activation and cytokine
Summary
Monocytes are white blood cells of the mononuclear phagocyte system, which derive from precursors in the bone marrow and circulate in the blood with a half-life of 1- 2 days before migrating into various tissues to replenish macrophages [1]. Blood monocytes are involved in the innate response to bacterial, fungal, parasitic, and viral infection [2]. While monocytes and macrophages were almost universally considered two related cell types that arise from a continuum of differentiation, recent studies have challenged this dogma’s generalized applicability based on three key findings. Monocytes do not substantially contribute to most tissue macrophage compartments in the steady-state or certain inflammation types. Mature tissue macrophages are derived from embryonic precursors that seed the tissues before birth. Tissue macrophages can maintain themselves in adults by self-renewal [3]
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