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A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues.
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 articleSubject
B-LymphocytesDendritic Cells
Basophils
Eosinophils
Neutrophils
Killer Cells, Natural
T-Lymphocytes
Macrophages
Mast Cells
Animals
Mice
Inflammation
Flow Cytometry
Cell Separation
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https://hdl.handle.net/10161/22234Published Version (Please cite this version)
10.1371/journal.pone.0150606Publication 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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Show full item recordScholars@Duke
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
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 unravel
Yen-Rei Andrea Yu
Adjunct Assistant Professor in the Department of Medicine
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