Current Protocols in Cytometry最新文献_第8页

Identification of Human Memory-Like NK Cells 人类记忆样NK细胞的鉴定

Current Protocols in Cytometry Pub Date : 2017-01-05 DOI: 10.1002/cpcy.13

Elena I. Kovalenko, Maria A. Streltsova, Leonid M. Kanevskiy, Sophia A. Erokhina, William G. Telford

{"title":"Identification of Human Memory-Like NK Cells","authors":"Elena I. Kovalenko, Maria A. Streltsova, Leonid M. Kanevskiy, Sophia A. Erokhina, William G. Telford","doi":"10.1002/cpcy.13","DOIUrl":"10.1002/cpcy.13","url":null,"abstract":"Our understanding of NK biology is increased dramatically, a product of improved flow-cytometric techniques for analyzing these cells. NK cells undergo significant changes in repertoire during differentiation. A repeating stimulus, such as a cytomegalovirus infection, may result in accumulation of certain types of highly differentiated NK cells designated as memory-like, or adaptive NK cells. Adaptive NK cells are capable of rapid expansion and effective response to the recall stimulus. These cells differ significantly from conventional NK cells both functionally and phenotypically. Here we describe an approach for identification and analysis of adaptive NK cells in human peripheral blood. CD57-positive cells with high expression of activating-receptor NKG2C, increased expression of KIR receptors, lack of co-expression with inhibitory receptor NKG2A, and decreased expression of activating receptor NCR3 (NKp30) all characterize this cell type. The flow-cytometric method described below can identify this NK cell subset on a relatively simple flow cytometer. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"79 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.13","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49504614","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

Measurement of T-Cell Telomere Length Using Amplified-Signal FISH Staining and Flow Cytometry 使用放大信号FISH染色和流式细胞术测量t细胞端粒长度

Current Protocols in Cytometry Pub Date : 2017-01-05 DOI: 10.1002/cpcy.11

Andrea L. Henning, Danielle E. Levitt, Jakob L. Vingren, Brian K. McFarlin

{"title":"Measurement of T-Cell Telomere Length Using Amplified-Signal FISH Staining and Flow Cytometry","authors":"Andrea L. Henning, Danielle E. Levitt, Jakob L. Vingren, Brian K. McFarlin","doi":"10.1002/cpcy.11","DOIUrl":"10.1002/cpcy.11","url":null,"abstract":"Exposure to pathogen-associated molecular patterns (PAMPS), damage-associated molecular patterns (DAMPS), and physiologically challenging stimuli either positively or negatively affect leukocyte maturity. Cellular maturity has implications for the effectiveness of host response to bacterial or viral infection and/or tissue injury. Thus, the ability to accurately assess cellular maturity and health is important to fully understand immune status and function. The most common technique for measuring cellular maturity is to measure telomere length; however, existing techniques are not optimized for single-cell measurements using flow cytometry. Specifically, existing methods used to measure telomere length are PCR-based, making it difficult for a researcher to measure maturity within specific leukocyte subsets (e.g., T cells). In this report, we describe a new approach for the measurement of telomere length within individual T cells using an amplified fluorescence in situ hybridization (FISH) technique (PrimeFlow RNA Assay). The unique aspect of this technique is that it amplifies the fluorescent signal rather than the target up to 3000-fold, resulting in the detection of as few as 1 copy of the target nucleic acid. While the current technique focuses on human T cells, this method can be broadly applied to a variety of cell types and disease models. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"79 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.11","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41970164","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

Quantitative Analysis of Cellular Senescence in Culture and In Vivo 细胞衰老在培养和体内的定量分析

Current Protocols in Cytometry Pub Date : 2017-01-05 DOI: 10.1002/cpcy.16

Jing Zhao, Heike Fuhrmann-Stroissnigg, Aditi U. Gurkar, Rafael R. Flores, Akaitz Dorronsoro, Donna B. Stolz, Claudette M. St. Croix, Laura J. Niedernhofer, Paul D. Robbins

{"title":"Quantitative Analysis of Cellular Senescence in Culture and In Vivo","authors":"Jing Zhao, Heike Fuhrmann-Stroissnigg, Aditi U. Gurkar, Rafael R. Flores, Akaitz Dorronsoro, Donna B. Stolz, Claudette M. St. Croix, Laura J. Niedernhofer, Paul D. Robbins","doi":"10.1002/cpcy.16","DOIUrl":"10.1002/cpcy.16","url":null,"abstract":"Cellular senescence refers to the irreversible growth arrest of normally dividing cells in response to various types of stress. Cellular senescence is induced by telomere shortening due to repeated cell division, which causes a DNA damage response, as well as genotoxic, oxidative, and inflammatory stress. Strong mitogenic signaling, such as oncogene activation, also drives cells into a senescent state. Senescent cells express a specific subset of genes, termed the senescence-associated secretory phenotype (SASP), including pro-inflammatory factors, growth factors, and matrix metalloproteinases, which together promote non-cell autonomous, secondary senescence. Clearance of senescent cells that accumulate with age improves health span, implicating cellular senescence as a contributing factor to the aging process. Thus, there is a need for methods to identify and quantify cellular senescence, both in cultured cells and in vivo. Here, methods for the most well-characterized and widely used senescent assays are described, from cell morphology and senescence-associated β-galactosidase (SA-βgal) staining to nuclear biomarkers, SASP, and altered levels of tumor suppressors. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"79 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.16","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49212898","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

Automated Measurement of Blood Vessels in Tissues from Microscopy Images 从显微图像中自动测量组织中的血管

Current Protocols in Cytometry Pub Date : 2016-10-10 DOI: 10.1002/cpcy.10

Neil J. Kelly, Nadine Dandachi, Dmitry A. Goncharov, Andressa Z. Pena, Josiah E. Radder, Alyssa D. Gregory, Yen-Chun Lai, Adriana S. Leme, Mark T. Gladwin, Elena A. Goncharova, Claudette M. St. Croix, Steven D. Shapiro

{"title":"Automated Measurement of Blood Vessels in Tissues from Microscopy Images","authors":"Neil J. Kelly, Nadine Dandachi, Dmitry A. Goncharov, Andressa Z. Pena, Josiah E. Radder, Alyssa D. Gregory, Yen-Chun Lai, Adriana S. Leme, Mark T. Gladwin, Elena A. Goncharova, Claudette M. St. Croix, Steven D. Shapiro","doi":"10.1002/cpcy.10","DOIUrl":"10.1002/cpcy.10","url":null,"abstract":"The quantification of tunica media thickness in histological cross sections is a ubiquitous exercise in cardiopulmonary research, yet the methods for quantifying medial wall thickness have never been rigorously examined with modern image analysis tools. As a result, inaccurate and cumbersome manual measurements of discrete wall regions along the vessel periphery have become common practice for wall thickness quantification. The aim of this study is to introduce, validate, and facilitate the use of an improved method for medial wall thickness quantification. We describe a novel method of wall thickness calculation based on image skeletonization and compare its results to those of common techniques. Using both theoretical and empirical approaches, we demonstrate the accuracy and superiority of the skeleton-based method for measuring wall thickness while discussing its interpretation and limitations. Finally, we present a new freely available software tool, the VMI Calculator, to facilitate wall thickness measurements using our novel method. © 2016 by John Wiley & Sons, Inc.","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"78 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2016-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.10","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50802932","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

Flow Analysis and Sorting of Plant Chromosomes 植物染色体的流动分析与分选

Current Protocols in Cytometry Pub Date : 2016-10-10 DOI: 10.1002/cpcy.9

Jan Vrána, Petr Cápal, Hana Šimková, Miroslava Karafiátová, Jana Čížková, Jaroslav Doležel

引用次数: 24

Whole Blood Analysis of Leukocyte-Platelet Aggregates 白细胞-血小板聚集体全血分析

Current Protocols in Cytometry Pub Date : 2016-10-10 DOI: 10.1002/cpcy.8

Anja J. Gerrits, Andrew L. Frelinger III, Alan D. Michelson

引用次数: 31

Measurement and Characterization of Apoptosis by Flow Cytometry 流式细胞术检测和表征细胞凋亡

Current Protocols in Cytometry Pub Date : 2016-07-01 DOI: 10.1002/cpcy.1

William Telford, Karen Tamul, Jolene Bradford

{"title":"Measurement and Characterization of Apoptosis by Flow Cytometry","authors":"William Telford, Karen Tamul, Jolene Bradford","doi":"10.1002/cpcy.1","DOIUrl":"10.1002/cpcy.1","url":null,"abstract":"Apoptosis is an important mechanism in cell biology, playing a critical regulatory role in virtually every organ system. It has been particularly well characterized in the immune system, with roles ranging from immature immune cell development and selection to down-regulation of the mature immune response. Apoptosis is also the primary mechanism of action of anti-cancer drugs. Flow cytometry has been the method of choice for analyzing apoptosis in suspension cells for more than 25 years. Numerous assays have been devised to measure both the earliest and latest steps in the apoptotic process, from the earliest signal-transduction events to the late morphological changes in cell shape and granularity, proteolysis, and chromatin condensation. These assays are particularly powerful when combined into multicolor assays determining several apoptotic characteristics simultaneously. The multiparametric nature of flow cytometry makes this technology particularly suited to measuring apoptosis. In this unit, we will describe the four main techniques for analyzing caspase activity in apoptotic cells, combined with annexin V and cell permeability analysis. These relatively simple multiparametric assays are powerful techniques for assessing cell death. © 2016 by John Wiley & Sons, Inc.","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"77 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2016-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.1","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34628552","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

A Quantitative Method for Comparing the Brightness of Antibody-dye Reagents and Estimating Antibodies Bound per Cell 一种比较抗体染色试剂亮度和估计每个细胞结合抗体的定量方法

Current Protocols in Cytometry Pub Date : 2016-07-01 DOI: 10.1002/cpcy.6

Aaron B. Kantor, Wayne A. Moore, Stephen Meehan, David R. Parks

{"title":"A Quantitative Method for Comparing the Brightness of Antibody-dye Reagents and Estimating Antibodies Bound per Cell","authors":"Aaron B. Kantor, Wayne A. Moore, Stephen Meehan, David R. Parks","doi":"10.1002/cpcy.6","DOIUrl":"10.1002/cpcy.6","url":null,"abstract":"We present a quantitative method for comparing the brightness of antibody-dye reagents and estimating antibodies bound per cell. The method is based on complementary binding of test and fill reagents to antibody capture microspheres. Several aliquots of antibody capture beads are stained with varying amounts of the test conjugate. The remaining binding sites on the beads are then filled with a second conjugate containing a different fluorophore. Finally, the fluorescence of the test conjugate compared to the fill conjugate is used to measure the relative brightness of the test conjugate. The fundamental assumption of the test-fill method is that if it takes X molecules of one test antibody to lower the fill signal by Y units, it will take the same X molecules of any other test antibody to give the same effect. We apply a quadratic fit to evaluate the test-fill signal relationship across different amounts of test reagent. If the fit is close to linear, we consider the test reagent to be suitable for quantitative evaluation of antibody binding. To calibrate the antibodies bound per bead, a PE conjugate with 1 PE molecule per antibody is used as a test reagent and the fluorescence scale is calibrated with Quantibrite PE beads. When the fluorescence per antibody molecule has been determined for a particular conjugate, that conjugate can be used for measurement of antibodies bound per cell. This provides comparisons of the brightness of different conjugates when conducted on an instrument whose statistical photoelectron (Spe) scales are known. © 2016 by John Wiley & Sons, Inc.","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"77 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2016-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.6","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34627967","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

High-Content Microscopy Analysis of Subcellular Structures: Assay Development and Application to Focal Adhesion Quantification 亚细胞结构的高含量显微分析:检测发展及其在黏附定量中的应用

Current Protocols in Cytometry Pub Date : 2016-07-01 DOI: 10.1002/cpcy.7

Torsten Kroll, David Schmidt, Georg Schwanitz, Mubashir Ahmad, Jana Hamann, Corinne Schlosser, Yu-Chieh Lin, Konrad J. Böhm, Jan Tuckermann, Aspasia Ploubidou

{"title":"High-Content Microscopy Analysis of Subcellular Structures: Assay Development and Application to Focal Adhesion Quantification","authors":"Torsten Kroll, David Schmidt, Georg Schwanitz, Mubashir Ahmad, Jana Hamann, Corinne Schlosser, Yu-Chieh Lin, Konrad J. Böhm, Jan Tuckermann, Aspasia Ploubidou","doi":"10.1002/cpcy.7","DOIUrl":"10.1002/cpcy.7","url":null,"abstract":"High-content analysis (HCA) converts raw light microscopy images to quantitative data through the automated extraction, multiparametric analysis, and classification of the relevant information content. Combined with automated high-throughput image acquisition, HCA applied to the screening of chemicals or RNAi-reagents is termed high-content screening (HCS). Its power in quantifying cell phenotypes makes HCA applicable also to routine microscopy. However, developing effective HCA and bioinformatic analysis pipelines for acquisition of biologically meaningful data in HCS is challenging. Here, the step-by-step development of an HCA assay protocol and an HCS bioinformatics analysis pipeline are described. The protocol's power is demonstrated by application to focal adhesion (FA) detection, quantitative analysis of multiple FA features, and functional annotation of signaling pathways regulating FA size, using primary data of a published RNAi screen. The assay and the underlying strategy are aimed at researchers performing microscopy-based quantitative analysis of subcellular features, on a small scale or in large HCS experiments. © 2016 by John Wiley & Sons, Inc.","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"77 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2016-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34627968","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

Measurement of Low‐Abundance Intracellular mRNA Using Amplified FISH Staining and Image‐Based Flow Cytometry 使用扩增的FISH染色和基于图像的流式细胞术测量细胞内低丰度mRNA

Current Protocols in Cytometry Pub Date : 2016-04-01 DOI: 10.1002/0471142956.cy0746s76

A. L. Henning, J. N. B. Sampson, B. McFarlin

{"title":"Measurement of Low‐Abundance Intracellular mRNA Using Amplified FISH Staining and Image‐Based Flow Cytometry","authors":"A. L. Henning, J. N. B. Sampson, B. McFarlin","doi":"10.1002/0471142956.cy0746s76","DOIUrl":"https://doi.org/10.1002/0471142956.cy0746s76","url":null,"abstract":"Recent advances in instrument design and reagent development have enabled the rapid progression in available measurement techniques in the field of flow cytometry. In particular, image‐based flow cytometry extends the analysis capacity found in traditional flow cytometry. Until recently, it was not possible to measure intracellular mRNA in specific phenotypes of cells by flow cytometry. In this protocol, a method of completing simultaneous intracellular measurement of mRNA and protein for PPAR‐gamma in peripheral blood monocytes, which have been exposed in vitro to modified LDL, is described. The process of PPAR‐gamma activation following uptake of modified LDL is believed to play a role in the development of atherogenesis. PPAR‐gamma mRNA measurement was made possible using an amplified FISH technique (PrimeFlow RNA Assay) that allowed for detection of low‐abundant intracellular mRNA expression. This protocol represents a continued effort by the authors’ laboratory to establish and validate new techniques to assess the role of the immune system in chronic disease. © 2016 by John Wiley & Sons, Inc.","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"46 3","pages":"7.46.1 - 7.46.8"},"PeriodicalIF":0.0,"publicationDate":"2016-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/0471142956.cy0746s76","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50822543","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