首页 > 最新文献

Current Protocols in Immunology最新文献

英文 中文
Issue Information TOC 发布信息TOC
Q2 Immunology and Microbiology Pub Date : 2019-09-03 DOI: 10.1002/cpim.67

Cover: In Geddes-McAlister and Gadjeva (https://doi.org/10.1002/cpim.87), Overview of workflow for MS-based proteomics profiling of neutrophils. Neutrophils are isolated and purified from bone marrow of 7- to 9-week-old male or female mice. The quality of the purification is assessed by FACS. Proteins are extracted by chemical (e.g., urea) and mechanical (e.g., sonication) cell disruption and enzymatically digested with Lys-C and trypsin proteases. The purified peptides are measured in the first MS scan and peptide fragmentation patterns are observed in the second MS scan (MS/;MS). The data is processed and analyzed using the publicly available MaxQuant and Perseus platforms. Figure generated with Biorender.com. See e87.

封面:在Geddes-McAlister和Gadjeva (https://doi.org/10.1002/cpim.87)中,中性粒细胞基于ms的蛋白质组学分析工作流程概述。中性粒细胞从7 ~ 9周龄的雄性或雌性小鼠骨髓中分离纯化。通过FACS对净化质量进行评估。通过化学(如尿素)和机械(如超声)破坏细胞提取蛋白质,并用赖氨酸- c和胰蛋白酶酶解。在第一次MS扫描中测量纯化的肽,并在第二次MS扫描中观察肽片段模式(MS/;MS)。使用公开的MaxQuant和Perseus平台对数据进行处理和分析。用Biorender.com生成的图。。英镑看到共有财产占有一席之地
{"title":"Issue Information TOC","authors":"","doi":"10.1002/cpim.67","DOIUrl":"10.1002/cpim.67","url":null,"abstract":"<p><b>Cover</b>: In Geddes-McAlister and Gadjeva (https://doi.org/10.1002/cpim.87), Overview of workflow for MS-based proteomics profiling of neutrophils. Neutrophils are isolated and purified from bone marrow of 7- to 9-week-old male or female mice. The quality of the purification is assessed by FACS. Proteins are extracted by chemical (e.g., urea) and mechanical (e.g., sonication) cell disruption and enzymatically digested with Lys-C and trypsin proteases. The purified peptides are measured in the first MS scan and peptide fragmentation patterns are observed in the second MS scan (MS/;MS). The data is processed and analyzed using the publicly available MaxQuant and Perseus platforms. Figure generated with Biorender.com. See e87.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"126 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.67","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42830613","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Experimental Mouse Model of Bleomycin-Induced Skin Fibrosis 博莱霉素致小鼠皮肤纤维化实验模型
Q2 Immunology and Microbiology Pub Date : 2019-08-27 DOI: 10.1002/cpim.88
Przemysław Błyszczuk, Anastasiia Kozlova, Zhongning Guo, Gabriela Kania, Oliver Distler

Systemic sclerosis (SSc) refers to an autoimmune disease, which is manifested by inflammation, vasculopathy, and fibrosis of the skin and internal organs. There are a number of different animal models recapitulating specific aspects of SSc. The experimental mouse model of bleomycin-induced skin fibrosis is commonly used to study the pathogenesis observed in SSc. In this model, repetitive intradermal injections of the cytotoxic agent bleomycin trigger progressive skin thickening, associated with excessive accumulation of collagen, infiltration of immune cells, and formation of α-smooth muscle actin (α-SMA)-positive myofibroblasts. In this article, we provide a detailed protocol for the induction of skin fibrosis in experimental mice by bleomycin. Moreover, we describe procedures for processing and analyzing affected skin tissue, provide troubleshooting, highlight advantages and limitations of the presented model, and critically discuss representative results. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Intradermal bleomycin injections to induce skin fibrosis in mice

Support Protocol: Mouse tissue collection for fibrosis evaluation and for other molecular assays

Basic Protocol 2: Evaluation of mouse skin thickness using Masson's trichrome staining

Basic Protocol 3: Measurement of hydroxyproline content in skin tissue using a colorimetric assay

Basic Protocol 4: Evaluation of myofibroblasts in mouse skin by immunohistochemistry

系统性硬化症(Systemic sclerosis, SSc)是一种自身免疫性疾病,主要表现为皮肤和内脏器官的炎症、血管病变和纤维化。有许多不同的动物模型概括了SSc的特定方面。博莱霉素致小鼠皮肤纤维化的实验模型是研究SSc发病机制的常用方法。在该模型中,反复皮内注射细胞毒性药物博来霉素引发皮肤进行性增厚,并伴有胶原蛋白的过度积累、免疫细胞的浸润和α-平滑肌肌动蛋白(α-SMA)阳性肌成纤维细胞的形成。在本文中,我们提供了一个详细的方案,以诱导皮肤纤维化的实验小鼠博来霉素。此外,我们描述了处理和分析受影响皮肤组织的程序,提供故障排除,突出所提出模型的优点和局限性,并批判性地讨论代表性结果。©2019 by John Wiley &基本方案1:皮内注射博来霉素诱导小鼠皮肤纤维化支持方案:收集小鼠组织用于纤维化评估和其他分子分析基本方案2:使用马松三色染色评估小鼠皮肤厚度基本方案3:使用比色法测量皮肤组织中羟丙氨酸含量基本方案4:使用免疫组织化学评估小鼠皮肤中的肌成纤维细胞
{"title":"Experimental Mouse Model of Bleomycin-Induced Skin Fibrosis","authors":"Przemysław Błyszczuk,&nbsp;Anastasiia Kozlova,&nbsp;Zhongning Guo,&nbsp;Gabriela Kania,&nbsp;Oliver Distler","doi":"10.1002/cpim.88","DOIUrl":"10.1002/cpim.88","url":null,"abstract":"<p>Systemic sclerosis (SSc) refers to an autoimmune disease, which is manifested by inflammation, vasculopathy, and fibrosis of the skin and internal organs. There are a number of different animal models recapitulating specific aspects of SSc. The experimental mouse model of bleomycin-induced skin fibrosis is commonly used to study the pathogenesis observed in SSc. In this model, repetitive intradermal injections of the cytotoxic agent bleomycin trigger progressive skin thickening, associated with excessive accumulation of collagen, infiltration of immune cells, and formation of α-smooth muscle actin (α-SMA)-positive myofibroblasts. In this article, we provide a detailed protocol for the induction of skin fibrosis in experimental mice by bleomycin. Moreover, we describe procedures for processing and analyzing affected skin tissue, provide troubleshooting, highlight advantages and limitations of the presented model, and critically discuss representative results. © 2019 by John Wiley &amp; Sons, Inc.</p><p><b>Basic Protocol 1</b>: Intradermal bleomycin injections to induce skin fibrosis in mice</p><p><b>Support Protocol</b>: Mouse tissue collection for fibrosis evaluation and for other molecular assays</p><p><b>Basic Protocol 2</b>: Evaluation of mouse skin thickness using Masson's trichrome staining</p><p><b>Basic Protocol 3</b>: Measurement of hydroxyproline content in skin tissue using a colorimetric assay</p><p><b>Basic Protocol 4</b>: Evaluation of myofibroblasts in mouse skin by immunohistochemistry</p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"126 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.88","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42484366","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 17
Mass Spectrometry-Based Quantitative Proteomics of Murine-Derived Polymorphonuclear Neutrophils 基于质谱的鼠源多形核中性粒细胞定量蛋白质组学研究
Q2 Immunology and Microbiology Pub Date : 2019-08-19 DOI: 10.1002/cpim.87
Jennifer Geddes-McAlister, Mihaela Gadjeva

Polymorphonuclear cells (PMNs or neutrophils) are the most abundant leukocyte in humans and represent an essential component of the innate immune system. The ability of neutrophils to initiate an immediate and non-specific host response against invading microbial species is the key to determining the outcome of infection. Neutrophils produce and secrete a plethora of immunomodulatory proteins, including major granule proteins and cytokines, as well as various enzymes, which regulate adherence, phagocytosis, chemotaxis, and cell survival. Historically, characterization of neutrophils and their roles during infection have relied on genetic and phenotypic analyses, as well as biochemical assays. However, recent advances in mass spectrometry-based proteomic workflows and technological platforms have supported the comprehensive profiling of neutrophil-associated immune responses in consideration of cellular factors and secreted proteins. Given the critical role of neutrophils in maintaining and regulating innate immune function, comprehensive profiling of their response to infection is imperative to ensuring host survival. Here, we briefly discuss the role of neutrophils in host-defense and describe methods to purify neutrophils from murine samples and comprehensively profile their proteomes. © 2019 by John Wiley & Sons, Inc.

多形核细胞(pmn或中性粒细胞)是人类最丰富的白细胞,是先天免疫系统的重要组成部分。中性粒细胞对入侵的微生物物种发起即时和非特异性宿主反应的能力是决定感染结果的关键。中性粒细胞产生和分泌大量的免疫调节蛋白,包括主要颗粒蛋白和细胞因子,以及各种调节粘附、吞噬、趋化和细胞存活的酶。从历史上看,中性粒细胞的特征及其在感染过程中的作用依赖于遗传和表型分析以及生化分析。然而,基于质谱的蛋白质组学工作流程和技术平台的最新进展支持了考虑细胞因子和分泌蛋白的中性粒细胞相关免疫反应的综合分析。鉴于中性粒细胞在维持和调节先天免疫功能中的关键作用,全面分析它们对感染的反应对于确保宿主生存是必要的。在这里,我们简要地讨论了中性粒细胞在宿主防御中的作用,并描述了从小鼠样本中纯化中性粒细胞并全面分析其蛋白质组的方法。©2019 by John Wiley &儿子,Inc。
{"title":"Mass Spectrometry-Based Quantitative Proteomics of Murine-Derived Polymorphonuclear Neutrophils","authors":"Jennifer Geddes-McAlister,&nbsp;Mihaela Gadjeva","doi":"10.1002/cpim.87","DOIUrl":"10.1002/cpim.87","url":null,"abstract":"<p>Polymorphonuclear cells (PMNs or neutrophils) are the most abundant leukocyte in humans and represent an essential component of the innate immune system. The ability of neutrophils to initiate an immediate and non-specific host response against invading microbial species is the key to determining the outcome of infection. Neutrophils produce and secrete a plethora of immunomodulatory proteins, including major granule proteins and cytokines, as well as various enzymes, which regulate adherence, phagocytosis, chemotaxis, and cell survival. Historically, characterization of neutrophils and their roles during infection have relied on genetic and phenotypic analyses, as well as biochemical assays. However, recent advances in mass spectrometry-based proteomic workflows and technological platforms have supported the comprehensive profiling of neutrophil-associated immune responses in consideration of cellular factors and secreted proteins. Given the critical role of neutrophils in maintaining and regulating innate immune function, comprehensive profiling of their response to infection is imperative to ensuring host survival. Here, we briefly discuss the role of neutrophils in host-defense and describe methods to purify neutrophils from murine samples and comprehensively profile their proteomes. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"126 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.87","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42027687","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 7
Production and Thermal Exchange of Conditional Peptide-MHC I Multimers 条件多肽- mhc I多聚体的制备和热交换
Q2 Immunology and Microbiology Pub Date : 2019-07-26 DOI: 10.1002/cpim.85
Jolien J. Luimstra, Kees L. M. C. Franken, Malgorzata A. Garstka, Jan W. Drijfhout, Jacques Neefjes, Huib Ovaa

Cytotoxic CD8+ T cells mediate cellular immunity through recognition of specific antigens presented by MHC class I on all nucleated cells. Studying T cell interactions and responses provides invaluable information on infection, autoimmunity and cancer. Fluorescently labeled multimers of MHC I can be used to quantify, characterize, and isolate specific CD8+ T cells by flow cytometry. Here we describe the production and use of conditional MHC I multimers that can be loaded with peptides of choice by incubating them at a defined temperature. Multimers are folded with a template peptide that forms a stable complex at low temperature, but dissociates at a defined elevated temperature. Using this technology multiple MHC I multimers can be generated in parallel, to allow staining and isolation of large sets of antigen-specific CD8+ T cells, especially when combined with barcoding technologies. © 2019 The Authors.

细胞毒性CD8+ T细胞通过识别MHC I类在所有有核细胞上呈递的特异性抗原介导细胞免疫。研究T细胞的相互作用和反应为感染、自身免疫和癌症提供了宝贵的信息。荧光标记的MHC I多聚体可用于定量,表征,并通过流式细胞术分离特异性CD8+ T细胞。在这里,我们描述了条件MHC I多聚体的生产和使用,这些多聚体可以通过在规定的温度下孵育而装载选择的肽。多聚体与模板肽折叠,模板肽在低温下形成稳定的复合物,但在特定的高温下解离。使用该技术,可以并行生成多个MHC I多聚体,以便对大量抗原特异性CD8+ T细胞进行染色和分离,特别是与条形码技术结合使用时。©2019作者。
{"title":"Production and Thermal Exchange of Conditional Peptide-MHC I Multimers","authors":"Jolien J. Luimstra,&nbsp;Kees L. M. C. Franken,&nbsp;Malgorzata A. Garstka,&nbsp;Jan W. Drijfhout,&nbsp;Jacques Neefjes,&nbsp;Huib Ovaa","doi":"10.1002/cpim.85","DOIUrl":"10.1002/cpim.85","url":null,"abstract":"<p>Cytotoxic CD8<sup>+</sup> T cells mediate cellular immunity through recognition of specific antigens presented by MHC class I on all nucleated cells. Studying T cell interactions and responses provides invaluable information on infection, autoimmunity and cancer. Fluorescently labeled multimers of MHC I can be used to quantify, characterize, and isolate specific CD8<sup>+</sup> T cells by flow cytometry. Here we describe the production and use of conditional MHC I multimers that can be loaded with peptides of choice by incubating them at a defined temperature. Multimers are folded with a template peptide that forms a stable complex at low temperature, but dissociates at a defined elevated temperature. Using this technology multiple MHC I multimers can be generated in parallel, to allow staining and isolation of large sets of antigen-specific CD8<sup>+</sup> T cells, especially when combined with barcoding technologies. © 2019 The Authors.</p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"126 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.85","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44912683","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 9
Characterization of Immune Cells from Adipose Tissue 脂肪组织免疫细胞的表征
Q2 Immunology and Microbiology Pub Date : 2019-07-24 DOI: 10.1002/cpim.86
Sagar P. Bapat, Yuqiong Liang, Ye Zheng

Adipose tissue (AT) serves a crucial role in maintaining organismal metabolic homeostasis. Studies have demonstrated that AT is populated with a diverse array of immune cells that coordinate and regulate AT function. This adipo-immune system is highly dynamic, reflecting the physiologic state of the organism (e.g., obese, lean, aged, or young) as well as the constant physiologic remodeling of AT associated with the daily rhythms of fasting and feeding. Many of the adaptive and maladaptive functional changes of AT are regulated by changes in the quantity and quality of distinct sets of AT-resident immune cells. Here we present protocols to assess the dynamic state of the immune system within AT by constructing censuses of adipose-resident immune cells (macrophages, dendritic cells, neutrophils, eosinophils, NK cells, innate lymphocytes, T cells, and B cells, etc.) based on flow cytometry, which we term adipo-immune profiles (AIPs). Constructing AIPs can be an integral part of assessment for AT health and function. This article describes the protocols to generate such AIPs. © 2019 by John Wiley & Sons, Inc.

脂肪组织(AT)在维持机体代谢稳态中起着至关重要的作用。研究表明,AT中存在多种协调和调节AT功能的免疫细胞。这种脂肪免疫系统是高度动态的,反映了生物体的生理状态(如肥胖、瘦弱、衰老或年轻),以及与禁食和喂养的日常节奏相关的AT的不断生理重塑。AT的许多适应性和非适应性功能变化是由不同AT驻留免疫细胞组的数量和质量变化调节的。在这里,我们提出了评估AT内免疫系统动态状态的方案,通过构建基于流式细胞术的脂肪驻留免疫细胞(巨噬细胞、树突状细胞、中性粒细胞、嗜酸性粒细胞、NK细胞、先天淋巴细胞、T细胞和B细胞等)普查,我们称之为脂肪免疫谱(AIPs)。构建AIPs可以成为评估AT健康和功能的一个组成部分。本文描述了生成此类aip的协议。©2019 by John Wiley &儿子,Inc。
{"title":"Characterization of Immune Cells from Adipose Tissue","authors":"Sagar P. Bapat,&nbsp;Yuqiong Liang,&nbsp;Ye Zheng","doi":"10.1002/cpim.86","DOIUrl":"10.1002/cpim.86","url":null,"abstract":"<p>Adipose tissue (AT) serves a crucial role in maintaining organismal metabolic homeostasis. Studies have demonstrated that AT is populated with a diverse array of immune cells that coordinate and regulate AT function. This adipo-immune system is highly dynamic, reflecting the physiologic state of the organism (e.g., obese, lean, aged, or young) as well as the constant physiologic remodeling of AT associated with the daily rhythms of fasting and feeding. Many of the adaptive and maladaptive functional changes of AT are regulated by changes in the quantity and quality of distinct sets of AT-resident immune cells. Here we present protocols to assess the dynamic state of the immune system within AT by constructing censuses of adipose-resident immune cells (macrophages, dendritic cells, neutrophils, eosinophils, NK cells, innate lymphocytes, T cells, and B cells, etc.) based on flow cytometry, which we term adipo-immune profiles (AIPs). Constructing AIPs can be an integral part of assessment for AT health and function. This article describes the protocols to generate such AIPs. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"126 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.86","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44221513","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 9
Using Evans Blue Dye to Determine Blood-Brain Barrier Integrity in Rodents 用埃文斯蓝染料测定啮齿动物血脑屏障完整性
Q2 Immunology and Microbiology Pub Date : 2019-07-19 DOI: 10.1002/cpim.83
Mariana Pereira de Souza Goldim, Amanda Della Giustina, Fabricia Petronilho

The blood-brain barrier (BBB) is an active and selective barrier that shields the brain from endogenous and exogenous insults. Different stimuli may lead to the disruption of this barrier, including inflammation and trauma. Several methods are used to evaluate BBB disruption. The most widely used method is Evans blue (EB) dye extravasation. EB cannot normally pass through the BBB and thus its presence in brain tissue indicates alterations in permeability. This protocol details the steps of EB extravasation in rodents. Important aspects regarding critical steps and advantages are also provided. © 2019 by John Wiley & Sons, Inc.

血脑屏障(BBB)是一种主动和选择性屏障,保护大脑免受内源性和外源性损伤。不同的刺激可能导致这一屏障的破坏,包括炎症和创伤。几种方法用于评估血脑屏障的破坏。应用最广泛的方法是埃文斯蓝(EB)染料外渗。EB通常不能通过血脑屏障,因此它在脑组织中的存在表明其渗透性的改变。本方案详细说明了EB在啮齿动物体内外渗的步骤。还提供了有关关键步骤和优势的重要方面。©2019 by John Wiley &儿子,Inc。
{"title":"Using Evans Blue Dye to Determine Blood-Brain Barrier Integrity in Rodents","authors":"Mariana Pereira de Souza Goldim,&nbsp;Amanda Della Giustina,&nbsp;Fabricia Petronilho","doi":"10.1002/cpim.83","DOIUrl":"10.1002/cpim.83","url":null,"abstract":"<p>The blood-brain barrier (BBB) is an active and selective barrier that shields the brain from endogenous and exogenous insults. Different stimuli may lead to the disruption of this barrier, including inflammation and trauma. Several methods are used to evaluate BBB disruption. The most widely used method is Evans blue (EB) dye extravasation. EB cannot normally pass through the BBB and thus its presence in brain tissue indicates alterations in permeability. This protocol details the steps of EB extravasation in rodents. Important aspects regarding critical steps and advantages are also provided. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"126 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.83","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45519510","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 73
Genome-Wide Measurement and Computational Analysis of Transcription Factor Binding and Chromatin Accessibility in Lymphocytes 淋巴细胞转录因子结合和染色质可及性的全基因组测量和计算分析
Q2 Immunology and Microbiology Pub Date : 2019-07-19 DOI: 10.1002/cpim.84
M. Firas Sadiyah, Rahul Roychoudhuri

Cells of the adaptive immune system, including CD4+ and CD8+ T cells, as well as B cells, possess the ability to undergo dynamic changes in population size, differentiation state, and function to counteract diverse and temporally stochastic threats from the external environment. To achieve this, lymphocytes must be able to rapidly control their gene-expression programs in a cell-type-specific manner and in response to extrinsic signals. Such capacity is provided by transcription factors (TFs), which bind to the available repertoire of regulatory DNA elements in distinct lymphocyte subsets to program cell-type-specific gene expression. Here we provide a set of protocols that utilize massively parallel sequencing–based approaches to map genome-wide TF-binding sites and accessible chromatin, with consideration of the unique aspects and technical issues facing their application to lymphocytes. We show how to computationally validate and analyze aligned data to map differentially enriched/accessible sites, identify enriched DNA sequence motifs, and detect the position of nucleosomes adjacent to accessible DNA elements. These techniques, when applied to immune cells, can enhance our understanding of how gene-expression programs are controlled within lymphocytes to coordinate immune function in homeostasis and disease. © 2019 by John Wiley & Sons, Inc.

适应性免疫系统的细胞,包括CD4+和CD8+ T细胞以及B细胞,具有群体大小、分化状态和功能动态变化的能力,以抵御来自外部环境的多样化和暂时随机的威胁。为了实现这一目标,淋巴细胞必须能够以细胞类型特异性的方式快速控制其基因表达程序,并响应外部信号。这种能力是由转录因子(TFs)提供的,它与不同淋巴细胞亚群中可用的调节性DNA元件结合,以编程细胞类型特异性基因表达。在这里,我们提供了一套方案,利用大规模并行测序的方法来绘制全基因组tf结合位点和可接近的染色质,考虑到它们在淋巴细胞中的应用所面临的独特方面和技术问题。我们展示了如何计算验证和分析对齐数据,以绘制差异富集/可访问的位点,识别富集的DNA序列基序,并检测核小体邻近可访问DNA元件的位置。当这些技术应用于免疫细胞时,可以增强我们对淋巴细胞内基因表达程序如何被控制以协调体内平衡和疾病中的免疫功能的理解。©2019 by John Wiley &儿子,Inc。
{"title":"Genome-Wide Measurement and Computational Analysis of Transcription Factor Binding and Chromatin Accessibility in Lymphocytes","authors":"M. Firas Sadiyah,&nbsp;Rahul Roychoudhuri","doi":"10.1002/cpim.84","DOIUrl":"10.1002/cpim.84","url":null,"abstract":"<p>Cells of the adaptive immune system, including CD4<sup>+</sup> and CD8<sup>+</sup> T cells, as well as B cells, possess the ability to undergo dynamic changes in population size, differentiation state, and function to counteract diverse and temporally stochastic threats from the external environment. To achieve this, lymphocytes must be able to rapidly control their gene-expression programs in a cell-type-specific manner and in response to extrinsic signals. Such capacity is provided by transcription factors (TFs), which bind to the available repertoire of regulatory DNA elements in distinct lymphocyte subsets to program cell-type-specific gene expression. Here we provide a set of protocols that utilize massively parallel sequencing–based approaches to map genome-wide TF-binding sites and accessible chromatin, with consideration of the unique aspects and technical issues facing their application to lymphocytes. We show how to computationally validate and analyze aligned data to map differentially enriched/accessible sites, identify enriched DNA sequence motifs, and detect the position of nucleosomes adjacent to accessible DNA elements. These techniques, when applied to immune cells, can enhance our understanding of how gene-expression programs are controlled within lymphocytes to coordinate immune function in homeostasis and disease. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"126 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.84","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43613099","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocols for the Analysis of microRNA Expression, Biogenesis, and Function in Immune Cells 免疫细胞中microRNA表达、生物发生和功能分析方案
Q2 Immunology and Microbiology Pub Date : 2019-06-20 DOI: 10.1002/cpim.78
Nannan Zhang, Guowu Hu, Timothy G. Myers, Peter R. Williamson

MicroRNAs (miRNAs) are short (19- to 25-nucleotide) noncoding RNA molecules that target mRNAs to repress gene expression and that play important roles in regulating many fundamental biological functions including cell differentiation, development, growth, and metabolism. They are well conserved in eukaryotic cells and are considered essential ancient elements of gene regulation. miRNA genes are transcribed by RNA polymerase II to generate primary miRNAs (pri-miRNAs), which are cleaved by microprocessor complex in the nucleus to generate stem-loop structures known as pre-miRNAs. Pre-miRNAs are translocated to the cytoplasm and cleaved by Dicer to form the mature miRNAs, which mediate mRNA degradation through their loading to the RNA-induced silencing complex (RISC) and binding to complementary sequences within target mRNAs to repress their translation by mRNA degradation and/or translation inhibition. Because ∼1900 miRNA genes are reported in the human genome, many associated with disease, appropriate methods to study miRNA expression and regulation under physiological and pathological conditions have become increasingly important to the study of many aspects of human biology, including immune regulation. As with small interfering RNA (siRNA), the mechanism of miRNA-mediated targeting has been used to develop miRNA-based therapeutics. For a complete and systematic analysis, it is critical to utilize a variety of different tools to analyze the expression of pri-mRNAs, pre-miRNAs, and mature miRNAs and characterize their targets both in vitro and in vivo. Such studies will facilitate future novel drug design and development. This unit provides six basic protocols for miRNA analysis, covering next-generation sequencing, quantitative real-time PCR (qRT-PCR), and digoxigenin-based expression analysis of pri-mRNAs, pre-miRNAs, and mature miRNAs; mapping of pri-miRNA and their cleavage sites by rapid amplification of cDNA ends (RACE); electrophoretic mobility shift assays (EMSAs) or biotin-based nonradioactive detection of miRNA-protein complexes (miRNPs); and functional analysis of miRNAs using miRNA mimics and inhibitors. © 2019 by John Wiley & Sons, Inc.

MicroRNAs (miRNAs)是一种短的(19- 25个核苷酸)非编码RNA分子,其靶向mrna抑制基因表达,在调节许多基本生物学功能(包括细胞分化、发育、生长和代谢)中发挥重要作用。它们在真核细胞中很好地保守,被认为是基因调控的基本古老元素。miRNA基因由RNA聚合酶II转录生成初级miRNA (pri-miRNA),由细胞核中的微处理器复合物切割产生茎环结构,称为pre-miRNA。pre - mirna易位到细胞质中,经Dicer切割形成成熟的mirna,通过装载到rna诱导沉默复合体(RISC)上,结合靶mRNA内的互补序列,通过mRNA降解和/或翻译抑制抑制靶mRNA的翻译,介导mRNA降解。由于在人类基因组中报道了约1900个miRNA基因,其中许多与疾病相关,因此研究生理和病理条件下miRNA表达和调控的适当方法对于研究人类生物学的许多方面(包括免疫调节)变得越来越重要。与小干扰RNA (siRNA)一样,mirna介导的靶向机制已被用于开发基于mirna的治疗方法。为了进行完整和系统的分析,利用各种不同的工具来分析pri- mrna、pre-miRNAs和成熟miRNAs的表达,并在体外和体内表征它们的靶点是至关重要的。这些研究将促进未来新药的设计和开发。该单元提供六种基本的miRNA分析方案,包括下一代测序、定量实时PCR (qRT-PCR)和基于地高辛的pri- mrna、前miRNAs和成熟miRNAs的表达分析;利用cDNA末端快速扩增(RACE)定位pri-miRNA及其切割位点;电泳迁移迁移试验(emsa)或基于生物素的mirna -蛋白复合物(miRNPs)的非放射性检测;以及使用miRNA模拟物和抑制剂对miRNA进行功能分析。©2019 by John Wiley &儿子,Inc。
{"title":"Protocols for the Analysis of microRNA Expression, Biogenesis, and Function in Immune Cells","authors":"Nannan Zhang,&nbsp;Guowu Hu,&nbsp;Timothy G. Myers,&nbsp;Peter R. Williamson","doi":"10.1002/cpim.78","DOIUrl":"10.1002/cpim.78","url":null,"abstract":"<p>MicroRNAs (miRNAs) are short (19- to 25-nucleotide) noncoding RNA molecules that target mRNAs to repress gene expression and that play important roles in regulating many fundamental biological functions including cell differentiation, development, growth, and metabolism. They are well conserved in eukaryotic cells and are considered essential ancient elements of gene regulation. miRNA genes are transcribed by RNA polymerase II to generate primary miRNAs (pri-miRNAs), which are cleaved by microprocessor complex in the nucleus to generate stem-loop structures known as pre-miRNAs. Pre-miRNAs are translocated to the cytoplasm and cleaved by Dicer to form the mature miRNAs, which mediate mRNA degradation through their loading to the RNA-induced silencing complex (RISC) and binding to complementary sequences within target mRNAs to repress their translation by mRNA degradation and/or translation inhibition. Because ∼1900 miRNA genes are reported in the human genome, many associated with disease, appropriate methods to study miRNA expression and regulation under physiological and pathological conditions have become increasingly important to the study of many aspects of human biology, including immune regulation. As with small interfering RNA (siRNA), the mechanism of miRNA-mediated targeting has been used to develop miRNA-based therapeutics. For a complete and systematic analysis, it is critical to utilize a variety of different tools to analyze the expression of pri-mRNAs, pre-miRNAs, and mature miRNAs and characterize their targets both <i>in vitro</i> and <i>in vivo</i>. Such studies will facilitate future novel drug design and development. This unit provides six basic protocols for miRNA analysis, covering next-generation sequencing, quantitative real-time PCR (qRT-PCR), and digoxigenin-based expression analysis of pri-mRNAs, pre-miRNAs, and mature miRNAs; mapping of pri-miRNA and their cleavage sites by rapid amplification of cDNA ends (RACE); electrophoretic mobility shift assays (EMSAs) or biotin-based nonradioactive detection of miRNA-protein complexes (miRNPs); and functional analysis of miRNAs using miRNA mimics and inhibitors. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"126 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.78","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47787406","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 17
Issue Information TOC 发布信息TOC
Q2 Immunology and Microbiology Pub Date : 2019-06-14 DOI: 10.1002/cpim.66

Cover: In Singh et al. (https://doi.org/10.1002/cpim.70), Hematoxylin- and eosin-stained sections of ear skin. Hematoxylin- and eosin-stained sections of untreated (left), rIL-23-treated (middle), and Aldara/IMQ-treated (right) ears. Acanthosis, thickening of the epidermis; hyperkeratosis, thickening of the stratum corneum; parakeratosis, retention of nucleated keratinocytes in the stratum corneum; micro-abscesses, neutrophil aggregates in the stratum corneum can be observed in these images. See e71.

封面:Singh等人(https://doi.org/10.1002/cpim.70),苏木精和伊红染色的耳朵皮肤切片。未处理(左)、ril -23处理(中)和Aldara/ imq处理(右)耳的苏木精和伊红染色切片。棘皮,表皮增厚;角化过度,角质层增厚;角化不全,有核角质细胞滞留在角质层;角质层可见微脓肿、中性粒细胞聚集。看到e71。
{"title":"Issue Information TOC","authors":"","doi":"10.1002/cpim.66","DOIUrl":"10.1002/cpim.66","url":null,"abstract":"<p><b>Cover</b>: In Singh et al. (https://doi.org/10.1002/cpim.70), Hematoxylin- and eosin-stained sections of ear skin. Hematoxylin- and eosin-stained sections of untreated (left), rIL-23-treated (middle), and Aldara/IMQ-treated (right) ears. Acanthosis, thickening of the epidermis; hyperkeratosis, thickening of the stratum corneum; parakeratosis, retention of nucleated keratinocytes in the stratum corneum; micro-abscesses, neutrophil aggregates in the stratum corneum can be observed in these images. See e71.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"125 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.66","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48109026","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
A Fast and Reliable Method to Isolate Human Placental Macrophages 一种快速可靠的分离人胎盘巨噬细胞的方法
Q2 Immunology and Microbiology Pub Date : 2019-05-24 DOI: 10.1002/cpim.77
Soraya Mezouar, Amira Ben Amara, Céline Chartier, Laurent Gorvel, Jean-Louis Mege

Macrophages are specialized cells involved in recognition, uptake, and destruction of microorganisms. Human placental macrophages are poorly investigated because of the lack of a convenient protocol for their isolation. Here, we present a straightforward and reliable method to isolate macrophages from full-term human placentas. After enzymatic digestion of placental tissue and centrifugation of the cell suspension on a Ficoll cushion, placental macrophages are selected using magnetic beads coated with anti-CD14 antibodies. Isolated cells are characterized by flow cytometry. Ninety eight percent of isolated CD14+ placental macrophages also express the macrophage marker CD68. Thus, this efficient and reliable method yields placental macrophages at high purity and sufficient quantity for functional studies. © 2019 by John Wiley & Sons, Inc.

巨噬细胞是参与识别、摄取和破坏微生物的特化细胞。人类胎盘巨噬细胞的研究很少,因为缺乏一种方便的分离方案。在这里,我们提出了一种直接可靠的方法从足月人胎盘中分离巨噬细胞。将胎盘组织酶解后,将细胞悬浮液在Ficoll缓冲垫上离心,用涂有抗cd14抗体的磁珠选择胎盘巨噬细胞。分离的细胞用流式细胞术进行鉴定。98%分离的CD14+胎盘巨噬细胞也表达巨噬细胞标志物CD68。因此,这种高效可靠的方法可以获得高纯度和足够数量的胎盘巨噬细胞,用于功能研究。©2019 by John Wiley &儿子,Inc。
{"title":"A Fast and Reliable Method to Isolate Human Placental Macrophages","authors":"Soraya Mezouar,&nbsp;Amira Ben Amara,&nbsp;Céline Chartier,&nbsp;Laurent Gorvel,&nbsp;Jean-Louis Mege","doi":"10.1002/cpim.77","DOIUrl":"10.1002/cpim.77","url":null,"abstract":"<p>Macrophages are specialized cells involved in recognition, uptake, and destruction of microorganisms. Human placental macrophages are poorly investigated because of the lack of a convenient protocol for their isolation. Here, we present a straightforward and reliable method to isolate macrophages from full-term human placentas. After enzymatic digestion of placental tissue and centrifugation of the cell suspension on a Ficoll cushion, placental macrophages are selected using magnetic beads coated with anti-CD14 antibodies. Isolated cells are characterized by flow cytometry. Ninety eight percent of isolated CD14<sup>+</sup> placental macrophages also express the macrophage marker CD68. Thus, this efficient and reliable method yields placental macrophages at high purity and sufficient quantity for functional studies. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"125 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.77","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37269119","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 10
期刊
Current Protocols in Immunology
全部 Acc. Chem. Res. ACS Applied Bio Materials ACS Appl. Electron. Mater. ACS Appl. Energy Mater. ACS Appl. Mater. Interfaces ACS Appl. Nano Mater. ACS Appl. Polym. Mater. ACS BIOMATER-SCI ENG ACS Catal. ACS Cent. Sci. ACS Chem. Biol. ACS Chemical Health & Safety ACS Chem. Neurosci. ACS Comb. Sci. ACS Earth Space Chem. ACS Energy Lett. ACS Infect. Dis. ACS Macro Lett. ACS Mater. Lett. ACS Med. Chem. Lett. ACS Nano ACS Omega ACS Photonics ACS Sens. ACS Sustainable Chem. Eng. ACS Synth. Biol. Anal. Chem. BIOCHEMISTRY-US Bioconjugate Chem. BIOMACROMOLECULES Chem. Res. Toxicol. Chem. Rev. Chem. Mater. CRYST GROWTH DES ENERG FUEL Environ. Sci. Technol. Environ. Sci. Technol. Lett. Eur. J. Inorg. Chem. IND ENG CHEM RES Inorg. Chem. J. Agric. Food. Chem. J. Chem. Eng. Data J. Chem. Educ. J. Chem. Inf. Model. J. Chem. Theory Comput. J. Med. Chem. J. Nat. Prod. J PROTEOME RES J. Am. Chem. Soc. LANGMUIR MACROMOLECULES Mol. Pharmaceutics Nano Lett. Org. Lett. ORG PROCESS RES DEV ORGANOMETALLICS J. Org. Chem. J. Phys. Chem. J. Phys. Chem. A J. Phys. Chem. B J. Phys. Chem. C J. Phys. Chem. Lett. Analyst Anal. Methods Biomater. Sci. Catal. Sci. Technol. Chem. Commun. Chem. Soc. Rev. CHEM EDUC RES PRACT CRYSTENGCOMM Dalton Trans. Energy Environ. Sci. ENVIRON SCI-NANO ENVIRON SCI-PROC IMP ENVIRON SCI-WAT RES Faraday Discuss. Food Funct. Green Chem. Inorg. Chem. Front. Integr. Biol. J. Anal. At. Spectrom. J. Mater. Chem. A J. Mater. Chem. B J. Mater. Chem. C Lab Chip Mater. Chem. Front. Mater. Horiz. MEDCHEMCOMM Metallomics Mol. Biosyst. Mol. Syst. Des. Eng. Nanoscale Nanoscale Horiz. Nat. Prod. Rep. New J. Chem. Org. Biomol. Chem. Org. Chem. Front. PHOTOCH PHOTOBIO SCI PCCP Polym. Chem.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1