小鼠组织中分离的中性粒细胞胞外陷阱的定量分析

Q1 Agricultural and Biological Sciences Current protocols in mouse biology Pub Date : 2020-07-23 DOI:10.1002/cpmo.78

Rebecca L. Buckland, Alicia S. Wilson, Anne Brüstle

{"title":"小鼠组织中分离的中性粒细胞胞外陷阱的定量分析","authors":"Rebecca L. Buckland, Alicia S. Wilson, Anne Brüstle","doi":"10.1002/cpmo.78","DOIUrl":null,"url":null,"abstract":"One of the most intriguing functions of neutrophils is the production of neutrophil extracellular traps (NETs), which are formed when neutrophils decondense their internal DNA and extrude it along with cytotoxic proteins in a web-like structure. This process allows neutrophils to trap and kill pathogens, and is also associated with multiple hematological and autoimmune conditions. Due to their rapid degradation, there are many challenges in accurately and specifically detecting and quantifying NETs. Microscopy is the gold standard for NET detection, but is not optimal for large-scale screening. Furthermore, methods relying on detection of free DNA or on flow cytometry–based examination of NET-associated markers can be nonspecific, time-consuming, and expensive. Here, we describe an innovative, quick, specific, and inexpensive conventional flow cytometry method for detecting neutrophils on the verge of forming NETs. These methods utilize pulse-shaped analysis (PulSA) to distinguish resting neutrophils from those with decondensed DNA, a prerequisite for NET formation. An increase in DNA-diffuse neutrophils is found in cell populations after exposure to NET-inducing stimuli, consistent with the DNA decondensation expected during neutrophil NET formation. These populations are only observed in granulocytes, validating the specificity of this method. We describe protocols optimized for neutrophils retrieved from mouse blood, spleen, and bone marrow. The relative speed and simplicity of the method described here makes it a useful tool for detecting NET formation in large-scale experiments. © 2020 Wiley Periodicals LLC.Basic Protocol: Detection of nuclear decondensation in neutrophils from stimulated murine bone marrowAlternate Protocol 1: Detection of nuclear decondensation in neutrophils from splenocytesAlternate Protocol 2: Detection of nuclear decondensation in neutrophils from bloodSupport Protocol 1: Cryopreservation and defrosting of samplesSupport Protocol 2: Paraformaldehyde fixation of samples","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2020-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.78","citationCount":"1","resultStr":"{\"title\":\"Quantification of Neutrophil Extracellular Traps Isolated From Mouse Tissues\",\"authors\":\"Rebecca L. Buckland, Alicia S. Wilson, Anne Brüstle\",\"doi\":\"10.1002/cpmo.78\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"One of the most intriguing functions of neutrophils is the production of neutrophil extracellular traps (NETs), which are formed when neutrophils decondense their internal DNA and extrude it along with cytotoxic proteins in a web-like structure. This process allows neutrophils to trap and kill pathogens, and is also associated with multiple hematological and autoimmune conditions. Due to their rapid degradation, there are many challenges in accurately and specifically detecting and quantifying NETs. Microscopy is the gold standard for NET detection, but is not optimal for large-scale screening. Furthermore, methods relying on detection of free DNA or on flow cytometry–based examination of NET-associated markers can be nonspecific, time-consuming, and expensive. Here, we describe an innovative, quick, specific, and inexpensive conventional flow cytometry method for detecting neutrophils on the verge of forming NETs. These methods utilize pulse-shaped analysis (PulSA) to distinguish resting neutrophils from those with decondensed DNA, a prerequisite for NET formation. An increase in DNA-diffuse neutrophils is found in cell populations after exposure to NET-inducing stimuli, consistent with the DNA decondensation expected during neutrophil NET formation. These populations are only observed in granulocytes, validating the specificity of this method. We describe protocols optimized for neutrophils retrieved from mouse blood, spleen, and bone marrow. The relative speed and simplicity of the method described here makes it a useful tool for detecting NET formation in large-scale experiments. © 2020 Wiley Periodicals LLC.Basic Protocol: Detection of nuclear decondensation in neutrophils from stimulated murine bone marrowAlternate Protocol 1: Detection of nuclear decondensation in neutrophils from splenocytesAlternate Protocol 2: Detection of nuclear decondensation in neutrophils from bloodSupport Protocol 1: Cryopreservation and defrosting of samplesSupport Protocol 2: Paraformaldehyde fixation of samples\",\"PeriodicalId\":37980,\"journal\":{\"name\":\"Current protocols in mouse biology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2020-07-23\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1002/cpmo.78\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Current protocols in mouse biology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/cpmo.78\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"Agricultural and Biological Sciences\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current protocols in mouse biology","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpmo.78","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"Agricultural and Biological Sciences","Score":null,"Total":0}

引用次数: 1

摘要

中性粒细胞最有趣的功能之一是产生中性粒细胞胞外陷阱(NETs)，当中性粒细胞去致密其内部DNA并将其与细胞毒性蛋白一起挤压成网状结构时形成。这个过程允许中性粒细胞捕获和杀死病原体，也与多种血液学和自身免疫性疾病有关。由于神经网络的快速降解，在准确、特异地检测和量化神经网络方面存在许多挑战。显微镜是NET检测的金标准，但不是大规模筛查的最佳选择。此外，依赖于游离DNA检测或基于流式细胞术的net相关标记物检测的方法可能是非特异性的，耗时且昂贵。在这里，我们描述了一种创新的、快速的、特异的、廉价的常规流式细胞术方法，用于检测处于形成NETs边缘的中性粒细胞。这些方法利用脉冲形分析(PulSA)来区分静止中性粒细胞和脱氧核糖核酸(NET形成的先决条件)。暴露于NET诱导刺激后，在细胞群中发现DNA弥漫性中性粒细胞增加，这与中性粒细胞NET形成过程中预期的DNA去浓缩一致。这些群体仅在粒细胞中观察到，验证了该方法的特异性。我们描述了从小鼠血液、脾脏和骨髓中提取中性粒细胞的优化方案。本文描述的方法的相对速度和简单性使其成为大规模实验中检测NET形成的有用工具。©2020 Wiley期刊有限公司基本方案:检测刺激小鼠骨髓中性粒细胞的核去浓缩替代方案1:检测脾细胞中性粒细胞的核去浓缩替代方案2:检测血液中性粒细胞的核去浓缩支持方案1:样品的冷冻保存和解冻支持方案2:样品的多聚甲醛固定

本文章由计算机程序翻译，如有差异，请以英文原文为准。

查看原文

微信好友朋友圈 QQ好友复制链接

本刊更多论文

Quantification of Neutrophil Extracellular Traps Isolated From Mouse Tissues

One of the most intriguing functions of neutrophils is the production of neutrophil extracellular traps (NETs), which are formed when neutrophils decondense their internal DNA and extrude it along with cytotoxic proteins in a web-like structure. This process allows neutrophils to trap and kill pathogens, and is also associated with multiple hematological and autoimmune conditions. Due to their rapid degradation, there are many challenges in accurately and specifically detecting and quantifying NETs. Microscopy is the gold standard for NET detection, but is not optimal for large-scale screening. Furthermore, methods relying on detection of free DNA or on flow cytometry–based examination of NET-associated markers can be nonspecific, time-consuming, and expensive. Here, we describe an innovative, quick, specific, and inexpensive conventional flow cytometry method for detecting neutrophils on the verge of forming NETs. These methods utilize pulse-shaped analysis (PulSA) to distinguish resting neutrophils from those with decondensed DNA, a prerequisite for NET formation. An increase in DNA-diffuse neutrophils is found in cell populations after exposure to NET-inducing stimuli, consistent with the DNA decondensation expected during neutrophil NET formation. These populations are only observed in granulocytes, validating the specificity of this method. We describe protocols optimized for neutrophils retrieved from mouse blood, spleen, and bone marrow. The relative speed and simplicity of the method described here makes it a useful tool for detecting NET formation in large-scale experiments. © 2020 Wiley Periodicals LLC.

Basic Protocol: Detection of nuclear decondensation in neutrophils from stimulated murine bone marrow

Alternate Protocol 1: Detection of nuclear decondensation in neutrophils from splenocytes

Alternate Protocol 2: Detection of nuclear decondensation in neutrophils from blood

Support Protocol 1: Cryopreservation and defrosting of samples

Support Protocol 2: Paraformaldehyde fixation of samples

求助全文

通过发布文献求助，成功后即可免费获取论文全文。去求助

来源期刊

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

自引率

0.00%

发文量

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.