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Current Protocols in Cytometry最新文献

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Flow Cytometry Instrumentation – An Overview 流式细胞仪仪器-概述
Current Protocols in Cytometry Pub Date : 2018-11-16 DOI: 10.1002/cpcy.52
Martin Büscher
{"title":"Flow Cytometry Instrumentation – An Overview","authors":"Martin Büscher","doi":"10.1002/cpcy.52","DOIUrl":"10.1002/cpcy.52","url":null,"abstract":"<p>The term flow cytometry, used since the seventies, describes a technology employed mainly in biology and medicine to measure and classify suspended particles, e.g., cells or microspheres. Measurable cell parameters include: geometric properties, such as cell size (diameter, surface area, volume); physiological properties (membrane potential, integrity, vitality); and quantities of DNA, RNA, cytokines, surface antigens, nuclear antigens, enzymes, and proteins. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"87 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-11-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.52","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36675749","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: 18
Flow Cytometry Method Validation Protocols 流式细胞术方法验证方案
Current Protocols in Cytometry Pub Date : 2018-11-12 DOI: 10.1002/cpcy.53
Nithianandan Selliah, Steven Eck, Cherie Green, Teri Oldaker, Jennifer Stewart, Alessandra Vitaliti, Virginia Litwin
{"title":"Flow Cytometry Method Validation Protocols","authors":"Nithianandan Selliah,&nbsp;Steven Eck,&nbsp;Cherie Green,&nbsp;Teri Oldaker,&nbsp;Jennifer Stewart,&nbsp;Alessandra Vitaliti,&nbsp;Virginia Litwin","doi":"10.1002/cpcy.53","DOIUrl":"10.1002/cpcy.53","url":null,"abstract":"<p>Analytical method validation provides a means to ensure that data are credible and reproducible. This unit will provide a brief introduction to analytical method validation as applied to cellular analysis by flow cytometry. In addition, the unit will provide practical procedures for three different types of validation. The first is a limited validation protocol that is applicable for research settings and non-regulated laboratories. The second is validation protocol that presents the minimum validation requirements in regulated laboratories. The third is a transfer validation protocol to be used when methods are transferred between laboratories. The recommendations presented in this unit are consistent with the white papers published by the American Association of Pharmaceutical Scientists and the International Clinical Cytometry Society, as well as with Clinical Laboratory Standards Institute Guideline H62: Validation of Assays Performed by Flow Cytometry (currently in preparation). © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"87 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.53","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36656832","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
Multiphoton Imaging of Collagen, Elastin, and Calcification in Intact Soft-Tissue Samples 完整软组织标本中胶原蛋白、弹性蛋白和钙化的多光子成像
Current Protocols in Cytometry Pub Date : 2018-10-31 DOI: 10.1002/cpcy.51
Piyusha S. Gade, Anne M. Robertson, Chih-Yuan Chuang
{"title":"Multiphoton Imaging of Collagen, Elastin, and Calcification in Intact Soft-Tissue Samples","authors":"Piyusha S. Gade,&nbsp;Anne M. Robertson,&nbsp;Chih-Yuan Chuang","doi":"10.1002/cpcy.51","DOIUrl":"10.1002/cpcy.51","url":null,"abstract":"<p>Multiphoton-induced second-harmonic generation and two-photon excitation enable imaging of collagen and elastin fibers at micron-level resolution to depths of hundreds of microns, without the use of exogenous stains. These attributes can be leveraged for quantitative analysis of the 3D architecture of collagen and elastin fibers within intact, soft tissue specimens such as the artery and bladder wall. This architecture influences the function of intramural cells and also plays a primary role in determining tissue passive mechanical properties. Calcification deposition in soft tissues is a highly prevalent pathology in both older and diseased populations that can alter tissue properties. In this unit, we provide a protocol for simultaneous multiphoton microscopy (MPM) imaging and analysis of 3D collagen and elastin structures with calcification, which is effective for fixed and fresh intact samples. We also provide an associated micro-CT protocol to identify regions of interest in the samples as a means to target the MPM imaging. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"87 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.51","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10264826","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: 13
Cell Cycle Analysis of Hematopoietic Stem and Progenitor Cells by Multicolor Flow Cytometry 多色流式细胞术分析造血干细胞和祖细胞的细胞周期
Current Protocols in Cytometry Pub Date : 2018-10-18 DOI: 10.1002/cpcy.50
Amy Galvin, Meredith Weglarz, Kat Folz-Donahue, Maris Handley, Misa Baum, Michael Mazzola, Hannah Litwa, David T. Scadden, Lev Silberstein
{"title":"Cell Cycle Analysis of Hematopoietic Stem and Progenitor Cells by Multicolor Flow Cytometry","authors":"Amy Galvin,&nbsp;Meredith Weglarz,&nbsp;Kat Folz-Donahue,&nbsp;Maris Handley,&nbsp;Misa Baum,&nbsp;Michael Mazzola,&nbsp;Hannah Litwa,&nbsp;David T. Scadden,&nbsp;Lev Silberstein","doi":"10.1002/cpcy.50","DOIUrl":"10.1002/cpcy.50","url":null,"abstract":"<p>Maintenance of hematopoietic stem cell (HSC) quiescence is critical for self-renewal and differentiation into mature lineages. Therefore, the ability to reliably detect abnormal HSC cycling is essential for experiments that seek to investigate abnormalities of HSC function. The ability to reproducibly evaluate cell cycle status in a rare cell subset requires careful optimization of multiple parameters during cell preparation and sample processing. Here, we describe a method where data acquisition parameters and fluorochrome combination for long-term HSC staining have been specifically designed for concurrent use with DAPI and Ki-67 antibodies. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"87 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.50","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36639032","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: 6
Issue Information TOC 发布信息TOC
Current Protocols in Cytometry Pub Date : 2018-10-01 DOI: 10.1002/cpcy.45
{"title":"Issue Information TOC","authors":"","doi":"10.1002/cpcy.45","DOIUrl":"10.1002/cpcy.45","url":null,"abstract":"","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"86 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.45","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47961223","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
Flow Cytometric Monitoring for Residual Disease in B Lymphoblastic Leukemia Post T Cell Engaging Targeted Therapies T细胞靶向治疗后B淋巴细胞白血病残留病变的流式细胞术监测
Current Protocols in Cytometry Pub Date : 2018-09-13 DOI: 10.1002/cpcy.44
Sindhu Cherian, Maryalice Stetler-Stevenson
{"title":"Flow Cytometric Monitoring for Residual Disease in B Lymphoblastic Leukemia Post T Cell Engaging Targeted Therapies","authors":"Sindhu Cherian,&nbsp;Maryalice Stetler-Stevenson","doi":"10.1002/cpcy.44","DOIUrl":"10.1002/cpcy.44","url":null,"abstract":"<p>The use of targeted therapy is growing in the setting of hematopoietic neoplasms. Flow cytometry is a cornerstone of residual disease monitoring post therapy in this group of malignancies. Often, there is overlap between antigens targeted by immunotherapies and gating reagents utilized for population identification by flow cytometry. Such overlap can render a previously excellent gating reagent inadequate for disease detection. Recently, several anti–CD19 T cell–engaging immunotherapeutic agents and an anti-CD22 immunotoxin have been FDA approved for use in B lymphoblastic leukemia (B-LL), with an anti–CD22 T cell–engaging agent in development. In the setting of such targeted therapies, CD19 and CD22 expression may be altered, compromising the use of these reagents for identification of abnormal blasts. We describe herein a strategy for flow cytometric monitoring for residual disease in patients with B-LL post T cell–engaging anti-CD19 and anti-CD22 therapies. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"86 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.44","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36489826","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: 28
Deriving Extracellular Vesicle Size From Scatter Intensities Measured by Flow Cytometry 从流式细胞术测量的散射强度中得出细胞外囊泡大小
Current Protocols in Cytometry Pub Date : 2018-08-31 DOI: 10.1002/cpcy.43
Leonie de Rond, Frank A. W. Coumans, Rienk Nieuwland, Ton G. van Leeuwen, Edwin van der Pol
{"title":"Deriving Extracellular Vesicle Size From Scatter Intensities Measured by Flow Cytometry","authors":"Leonie de Rond,&nbsp;Frank A. W. Coumans,&nbsp;Rienk Nieuwland,&nbsp;Ton G. van Leeuwen,&nbsp;Edwin van der Pol","doi":"10.1002/cpcy.43","DOIUrl":"10.1002/cpcy.43","url":null,"abstract":"<p>Flow cytometry is commonly used to investigate the potential for extracellular vesicles (EVs) to be biomarkers of disease. A typical flow cytometer detects fluorescence and scatter intensities of single EVs in arbitrary units. These arbitrary units complicate data interpretation and data comparison between different flow cytometers. For example, comparison of detected EV concentrations requires knowledge of the detectable EV sizes. Using Mie theory and knowledge of the optical configuration of the flow cytometer, EV size can be derived from the scatter intensity for a given EV refractive index. Here, a protocol is described to derive the size of EVs and other nanoparticles from the scatter intensity. The resulting size distribution allows the comparison of data between flow cytometers, which is a prerequisite for clinical application of EVs as biomarkers and may advance other fields where sizing of nanoparticles is essential. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"86 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.43","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36450591","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: 41
Clearing for Deep Tissue Imaging 深层组织成像清理
Current Protocols in Cytometry Pub Date : 2018-07-13 DOI: 10.1002/cpcy.38
Michael Muntifering, Daniel Castranova, Gregory A. Gibson, Evan Meyer, Matthew Kofron, Alan M. Watson
{"title":"Clearing for Deep Tissue Imaging","authors":"Michael Muntifering,&nbsp;Daniel Castranova,&nbsp;Gregory A. Gibson,&nbsp;Evan Meyer,&nbsp;Matthew Kofron,&nbsp;Alan M. Watson","doi":"10.1002/cpcy.38","DOIUrl":"10.1002/cpcy.38","url":null,"abstract":"<p>Biologic tissues are generally opaque due to optical properties that result in scattering and absorption of light. Preparation of tissues for optical microscopy often involves sectioning to a thickness of 50-100 µm, the practical limits of light penetration and recovery. A researcher who wishes to image a whole tissue must acquire potentially hundreds of individual sections before rendering them into a three-dimensional volume. Clearing removes strongly light-scattering and light-absorbing components of a tissue and equalizes the refractive index of the imaging medium to that of the tissue. After clearing, the maximum depth of imaging is often defined by the microscope optics rather than the tissue. Such visibility enables the interrogation of whole tissues and even animals without the need to section. Researchers can study a biological process in the context of its three-dimensional environment, identify rare events in large volumes of tissues, and trace cells and cell-cell interactions over large distances. This article describes four popular clearing protocols that are relevant to a wide variety of scenarios across biologic disciplines: CUBIC, CLARITY, 3DISCO, and SeeDB. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"86 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-07-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.38","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36309713","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: 28
Resolution of Viable and Membrane-Compromised Free Bacteria in Aquatic Environments by Flow Cytometry 用流式细胞术分离水生环境中活菌和膜受损的游离细菌
Current Protocols in Cytometry Pub Date : 2018-06-29 DOI: 10.1002/cpcy.42
Gérald Grégori, Michel Denis, Sergio Sgorbati, Sandra Citterio
{"title":"Resolution of Viable and Membrane-Compromised Free Bacteria in Aquatic Environments by Flow Cytometry","authors":"Gérald Grégori,&nbsp;Michel Denis,&nbsp;Sergio Sgorbati,&nbsp;Sandra Citterio","doi":"10.1002/cpcy.42","DOIUrl":"10.1002/cpcy.42","url":null,"abstract":"<p>In aquatic environments, free heterotrophic bacteria play an extremely important role due to their high biomass, wide panel of metabolisms, and ubiquity, as well as the toxicity of certain species. This unit presents a nucleic-acid double-staining protocol (NADS) for flow cytometry that can distinguish fractions of viable, damaged, or membrane-compromised cells within the free-bacterial community. The NADS protocol is based on the simultaneous utilization of two nucleic acid stains—membrane-permeant SYBR Green and membrane-impermeant propidium iodide (PI). The efficiency of the double staining on fresh samples is magnified by the FRET from SYBR Green to PI when both are bound to the nucleic acids. Full quenching of SYBR Green fluorescence by PI identifies cells with a compromised membrane, partial quenching indicates cells with a slightly damaged membrane, and lack of quenching characterizes cells with an intact membrane. Samples do not require any pretreatment and this protocol can be performed almost anywhere. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"85 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.42","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36270589","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: 4
Mitochondrial Subtype Identification and Characterization 线粒体亚型鉴定与表征
Current Protocols in Cytometry Pub Date : 2018-06-26 DOI: 10.1002/cpcy.41
Joseph R. Daniele, Kartoosh Heydari, Andrew Dillin
{"title":"Mitochondrial Subtype Identification and Characterization","authors":"Joseph R. Daniele,&nbsp;Kartoosh Heydari,&nbsp;Andrew Dillin","doi":"10.1002/cpcy.41","DOIUrl":"10.1002/cpcy.41","url":null,"abstract":"<p>Healthy, functional mitochondria are central to many cellular and physiological phenomena, including aging, metabolism, and stress resistance. A key feature of healthy mitochondria is a high membrane potential (Δψ) or charge differential (i.e., proton gradient) between the matrix and inner mitochondrial membrane. Mitochondrial Δψ has been extensively characterized via flow cytometry of intact cells, which measures the average membrane potential within a cell. However, the characteristics of individual mitochondria differ dramatically even within a single cell, and thus interrogation of mitochondrial features at the organelle level is necessary to better understand and accurately measure heterogeneity. Here we describe a new flow cytometric methodology that enables the quantification and classification of mitochondrial subtypes (via their Δψ, size, and substructure) using the small animal model <i>C. elegans</i>. Future application of this methodology should allow research to discern the bioenergetic and mitochondrial component in a number of human disease and aging models, including, <i>C. elegans</i>, cultured cells, small animal models, and human biopsy samples. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"85 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.41","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36259061","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: 1
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