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Heterogeneity of Human Monocytes: An Optimized Four-Color Flow Cytometry Protocol for Analysis of Monocyte Subsets

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Abstract

Monocytes are central mediators in the development of atherosclerotic plaques. They circulate in blood and eventually migrate into tissue including the vessel wall where they give rise to macrophages and dendritic cells. The existence of monocyte subsets with distinct roles in homeostasis and inflammation suggests specialization of function. These subsets are identified based on expression of the CD14 and CD16 markers. Routinely applicable protocols remain elusive, however. Here, we present an optimized four-color flow cytometry protocol for analysis of human blood monocyte subsets using a specific PE-Cy5–conjugated monoclonal antibody (mAb) to HLA-DR, a PE-Cy7-conjugated mAb to CD14, a FITC-conjugated mAb to CD16, and PE-conjugated mAbs to additional markers relevant to monocyte function. Classical CD14+CD16 monocytes (here termed “Mo1” subset) expressed high CCR2, CD36, CD64, and CD62L, but low CX3CR1, whereas “nonclassical” CD14loCD16+ monocytes (Mo3) essentially showed the inverse expression pattern. CD14+CD16+ monocytes (Mo2) expressed high HLA-DR, CD36, and CD64. In patients with stable coronary artery disease (n = 13), classical monocytes were decreased, whereas “nonclassical” monocytes were increased 90% compared with healthy subjects with angiographically normal coronary arteries (n = 14). Classical monocytes from CAD patients expressed higher CX3CR1 and CCR2 than controls. Thus, stable CAD is associated with expansion of the nonclassical monocyte subset and increased expression of inflammatory markers on monocytes. Flow cytometric analysis of monocyte subsets and marker expression may provide valuable information on vascular inflammation. This may translate into the identification of monocyte subsets as selective therapeutic targets, thus avoiding adverse events associated with indiscriminate monocyte inhibition.

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Acknowledgments

Support by the Swiss Cardiology Foundation, Cardiomet-Cardiovascular Pole of the Faculty of Biology and Medicine of the University of Lausanne, and the Fondazione Cecilia Augusta is greatly appreciated.

Expert advice by Dr. Christopher Kornfeld (Application Specialist Flow Cytometry, Beckman Coulter) and Dr. Jorge Formaro (Application Specialist Flow Cytometry, Instrumentation Laboratory) regarding flow cytometric protocols was greatly appreciated.

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Correspondence to Tiziano Tallone or Giuseppe Vassalli.

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Tallone, T., Turconi, G., Soldati, G. et al. Heterogeneity of Human Monocytes: An Optimized Four-Color Flow Cytometry Protocol for Analysis of Monocyte Subsets. J. of Cardiovasc. Trans. Res. 4, 211–219 (2011). https://doi.org/10.1007/s12265-011-9256-4

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  • DOI: https://doi.org/10.1007/s12265-011-9256-4

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