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One approach commonly used to examine immune cells in non-lymphoid tissues is to focus on a limited number of cell types. For this reason, these protocols display a number of characteristic limitations. Currently described protocols for the flow cytometric analysis of non-lymphoid tissues represent a compromise between maximizing the number of informative cell surface markers examined and using a limited number of fluorescent channels. Unfortunately, no such method has yet been described. Ideally, a method for performing flow cytometric analysis would be available that is both comprehensive and widely applicable capable of accurately identifying and quantifying all immune cell types in almost any tissue, either at baseline or during an inflammatory response. Flow cytometric analysis furnishes important insights into the immune status of a given tissue by providing information about the numbers and phenotypes of the immune cells that the tissue contains. The work is made available under the Creative Commons CC0 public domain dedication.ĭata Availability: All relevant data are within the paper and its Supporting Information files.įunding: This work was supported by a Pulmonary Hypertension Association Proof of Concept Award and Mandel Foundation Fellowship award.Ĭompeting interests: The authors have declared that no competing interests exist.įlow cytometry is used extensively to examine immune cell repertoires and follow immune responses in non-lymphoid tissues.
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This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Received: AugAccepted: FebruPublished: March 3, 2016 Mueller, The University of Melbourne, AUSTRALIA (2016) A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.Ĭitation: Yu Y-RA, O’Koren EG, Hotten DF, Kan MJ, Kopin D, Nelson ER, et al. 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 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. We also characterized the expression patterns of several commonly used myeloid and macrophage markers.
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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 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. 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. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues.