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A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues.

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Date
2016-01
Authors
Yu, Yen-Rei A
O'Koren, Emily G
Hotten, Danielle F
Kan, Matthew J
Kopin, David
Nelson, Erik R
Que, Loretta
Gunn, Michael D
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Abstract
Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.
Type
Journal article
Subject
B-Lymphocytes
Dendritic Cells
Basophils
Eosinophils
Neutrophils
Killer Cells, Natural
T-Lymphocytes
Macrophages
Mast Cells
Animals
Mice
Inflammation
Flow Cytometry
Cell Separation
Permalink
https://hdl.handle.net/10161/22234
Published Version (Please cite this version)
10.1371/journal.pone.0150606
Publication Info
Yu, Yen-Rei A; O'Koren, Emily G; Hotten, Danielle F; Kan, Matthew J; Kopin, David; Nelson, Erik R; ... Gunn, Michael D (2016). A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues. PloS one, 11(3). pp. e0150606. 10.1371/journal.pone.0150606. Retrieved from https://hdl.handle.net/10161/22234.
This is constructed from limited available data and may be imprecise. To cite this article, please review & use the official citation provided by the journal.
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Scholars@Duke

Gunn

Michael Dee Gunn

Professor of Medicine
The focus of my work is on understanding how dendritic cells, monocytes, and macrophages regulate immune responses, contribute to specific disease pathologies, and can be manipulated to stimulate or inhibit specific immune responses. We are also using our knowledge of immunology to develop diagnostics and therapeutics for a variety of human diseases.  Lab History The lab started with our discovery of the lymphoid chemokines, which regulate the mi
Que

Loretta Georgina Que

Professor of Medicine
My research interests focus on studying the role of nitric oxide and related enzymes in the pathogenesis of lung disease, specifically that caused by nitrosative/oxidative stress. Proposed studies are performed in cell culture and applied to animal models of disease, then examined in human disease where relevant. It is our hope that by better understanding the role of NO and reactive nitrogen species in mediating inflammation, and regulating cell signaling, that we will not only help to unr
Yu

Yen-Rei Andrea Yu

Adjunct Assistant Professor in the Department of Medicine
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