一种快速、简便、可定制的八色流式细胞术方法用于分析小鼠支气管肺泡灌洗液的细胞含量

Q1 Agricultural and Biological Sciences

Current protocols in mouse biology Pub Date : 2017-06-19 DOI:10.1002/cpmo.26

François Daubeuf, Julien Becker, Juan Antonio Aguilar-Pimentel, Claudine Ebel, Martin Hrabě de Angelis, Yann Hérault, Nelly Frossard

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引用次数: 5

摘要

支气管肺泡灌洗液(BAL)的细胞组成是气道炎症的重要指标。通常是在载玻片上对白细胞进行细胞离心，然后在光镜下根据形态学标准对其进行染色、鉴定和计数，分别为嗜酸性粒细胞、中性粒细胞、巨噬细胞或淋巴细胞，其中巨噬细胞和淋巴细胞并不总是容易区分。我们在这里描述了一步，易于使用，易于定制的8色流式细胞术方法，用于进行差异细胞计数，并将其与染色细胞自旋的形态学计数进行比较。该方法通过使用抗体识别T细胞、B细胞、中性粒细胞、嗜酸性粒细胞和巨噬细胞的同时一步免疫标记程序来识别BAL细胞。流式分选细胞子集的形态学分析用于验证该协议。这种基本流式细胞术方案的一个重要优点是能够通过添加抗体来定制它，以研究白细胞细胞表面的受体表达，并根据需要识别炎症细胞的亚类。©2017 by John Wiley &儿子,Inc。

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A Fast, Easy, and Customizable Eight-Color Flow Cytometric Method for Analysis of the Cellular Content of Bronchoalveolar Lavage Fluid in the Mouse

The cell composition of bronchoalveolar lavage fluid (BAL) is an important indicator of airway inflammation. It is commonly determined by cytocentrifuging leukocytes on slides, then staining, identifying, and counting them as eosinophils, neutrophils, macrophages, or lymphocytes according to morphological criteria under light microscopy, where it is not always easy to distinguish macrophages from lymphocytes. We describe here a one-step, easy-to-use, and easy-to-customize 8-color flow cytometric method for performing differential cell count and comparing it to morphological counts on stained cytospins. This method identifies BAL cells by a simultaneous one-step immunolabeling procedure using antibodies to identify T cells, B cells, neutrophils, eosinophils, and macrophages. Morphological analysis of flow-sorted cell subsets is used to validate this protocol. An important advantage of this basic flow cytometry protocol is the ability to customize it by the addition of antibodies to study receptor expression at leukocyte cell surfaces and identify subclasses of inflammatory cells as needed. © 2017 by John Wiley & Sons, Inc.

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来源期刊

Current protocols in mouse biology Agricultural and Biological Sciences-Animal Science and Zoology

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet, all too often, nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. The aim of Current Protocols in Mouse Biology is to provide the clearest, most detailed and reliable step-by-step instructions for protocols involving the use of mice in biomedical research. Written by experts in the field and extensively edited to our exacting standards, the protocols include all of the information necessary to complete an experiment in the laboratory—introduction, materials lists with supplier information, detailed step-by-step procedures with helpful annotations, recipes for reagents and solutions, illustrative figures and information-packed tables. Each article also provides invaluable discussions of background information, applications of the methods, important assumptions, key parameters, time considerations, and tips to help avoid common pitfalls and troubleshoot experiments. Furthermore, Current Protocols in Mouse Biology content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Mouse Biology brings together resources in mouse biology and genetics and provides a mouse protocol resource that covers all aspects of mouse biology. Current Protocols in Mouse Biology also permits optimization of mouse model usage, which is significantly impacted by both cost and ethical constraints. Optimal and standardized mouse protocols ultimately reduce experimental variability and reduce the number of animals used in mouse experiments.