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.

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Citation

Published Version (Please cite this version)

10.1371/journal.pone.0150606

Publication Info

Yu, Yen-Rei A, Emily G O'Koren, Danielle F Hotten, Matthew J Kan, David Kopin, Erik R Nelson, Loretta Que, Michael D Gunn, et al. (2016). A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues. PloS one, 11(3). p. e0150606. 10.1371/journal.pone.0150606 Retrieved from https://hdl.handle.net/10161/22234.

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Scholars@Duke

Yu

Yen-Rei Andrea Yu

Adjunct Assistant Professor in the Department of Medicine
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 unravel the basic mechanisms of NO related lung disease, but also provide a rationale for targeted therapeutic use of NO.


Key words: nitrosative defense, lung injury, nitric oxide

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 migration of lymphocytes and dendritic cells to and within secondary lymphoid organs.  We identified the chemokine (CCL21) that mediates the entry of naïve T cells and activated dendritic cells into lymph nodes and the chemokine (CXCL13) that mediates the entry of B cells into lymphoid follicles.  Our focus then shifted to understanding how specific cell types, primarily dendritic cells, and cell migration events regulate immune responses.  We identified murine plasmacytoid dendritic cells; the cell type that causes pulmonary immune pathology during influenza infection; the dendritic cell type that stimulates Th1 immune responses; the cell type that induces neuronal injury in Alzheimer's disease, and the macrophage type that stimulates pulmonary hypertension.  Our current work continues these basic studies while applying our findings to models of human disease. 

Current Research 

Tumor immune therapeutics – We have developed a novel cellular vaccine strategy for the treatment of cancer.  This strategy is much simpler, more cost effective, more clinically feasible, and much more efficacious than classic dendritic cell vaccines.  We are now determining the mechanisms by which this vaccine induces such potent immune responses and advancing it to initial human clinical trials.

Development of recombinant antibodies as diagnostic reagents – Our lab has developed novel methods to generate recombinant single chain antibodies using phage display technology.  We are currently using these methods to generate pathogen-specific antibodies for use in diagnostic tests for a variety of human bacterial, viral, and fungal infections.  In collaboration with Duke Biomedical Engineering, we are deploying our antibodies in a novel diagnostic assay platform to develop point-of-care assays for the diagnosis of a variety of emerging pathogens.  Our recently developed point-of-care assay for Ebola virus displays a sensitivity superior to PCR at a fraction of the per assay cost.


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