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Identification of Human Memory-Like NK Cells 人类记忆样NK细胞的鉴定
Q1 Health Professions 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

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.

我们对NK生物学的理解急剧增加,这是用于分析这些细胞的改进的流式细胞术技术的产物。NK细胞在分化过程中发生了显著的库变化。重复刺激,如巨细胞病毒感染,可能导致某些类型的高度分化NK细胞的积累,称为记忆样NK细胞或适应性NK细胞。适应性NK细胞对回忆刺激具有快速扩增和有效反应的能力。这些细胞在功能和表型上都与传统NK细胞有很大的不同。在这里,我们描述了一种方法来鉴定和分析适应性NK细胞在人外周血。cd57阳性细胞具有活化受体NKG2C高表达、KIR受体表达增加、缺乏与抑制受体NKG2A共表达、活化受体NCR3 (NKp30)表达降低等特征。下面描述的流式细胞术方法可以在一个相对简单的流式细胞仪上识别NK细胞亚群。©2017 by John Wiley &儿子,Inc。
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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
Q1 Health Professions Pub Date : 2017-01-05 DOI: 10.1002/cpcy.11
Andrea L. Henning, Danielle E. Levitt, Jakob L. Vingren, Brian K. McFarlin

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.

暴露于病原体相关分子模式(PAMPS)、损伤相关分子模式(DAMPS)和生理上具有挑战性的刺激会对白细胞成熟度产生积极或消极的影响。细胞成熟度影响宿主对细菌或病毒感染和/或组织损伤反应的有效性。因此,准确评估细胞成熟度和健康状况的能力对于充分了解免疫状态和功能非常重要。测量细胞成熟度最常用的技术是测量端粒长度;然而,现有的技术并没有优化单细胞测量使用流式细胞术。具体来说,用于测量端粒长度的现有方法是基于pcr的,这使得研究人员难以测量特定白细胞亚群(例如T细胞)的成熟度。在本报告中,我们描述了一种使用扩增荧光原位杂交(FISH)技术(PrimeFlow RNA Assay)测量单个T细胞端粒长度的新方法。这项技术的独特之处在于,它将荧光信号而不是目标放大了3000倍,从而可以检测到目标核酸的1个拷贝。虽然目前的技术主要集中在人类T细胞上,但这种方法可以广泛应用于各种细胞类型和疾病模型。©2017 by John Wiley &儿子,Inc。
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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
Q1 Health Professions 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

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.

细胞衰老是指正常分裂细胞在各种应激作用下的不可逆生长停滞。细胞衰老是由重复细胞分裂引起的端粒缩短引起的,这会引起DNA损伤反应,以及基因毒性、氧化和炎症应激。强烈的有丝分裂信号,如癌基因激活,也会驱使细胞进入衰老状态。衰老细胞表达一种特定的基因亚群,称为衰老相关分泌表型(SASP),包括促炎因子、生长因子和基质金属蛋白酶,它们共同促进非细胞自主的继发性衰老。清除随着年龄增长而积累的衰老细胞可以改善健康寿命,这表明细胞衰老是衰老过程的一个促成因素。因此,需要一种方法来鉴定和量化细胞衰老,无论是在培养细胞还是在体内。本文描述了最具特征和广泛使用的衰老测定方法,从细胞形态学和衰老相关的β-半乳糖苷酶(SA-βgal)染色到核生物标志物、SASP和肿瘤抑制因子水平的改变。©2017 by John Wiley &儿子,Inc。
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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
Q1 Health Professions 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

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.

在心肺研究中,组织学横断面中中膜厚度的定量是一项普遍的工作,但量化内壁厚度的方法从未被现代图像分析工具严格检验过。因此,沿着血管外围的离散壁区进行不准确和繁琐的人工测量已成为壁厚量化的常见做法。本研究的目的是介绍、验证和促进一种改进的内侧壁厚度定量方法的使用。我们描述了一种基于图像骨架化的壁厚计算新方法,并将其结果与常用技术的结果进行了比较。使用理论和经验方法,我们证明了基于骨架的方法测量壁厚的准确性和优越性,同时讨论了其解释和局限性。最后,我们提出了一个新的免费软件工具,VMI计算器,以方便使用我们的新方法测量壁厚。©2016 by John Wiley &儿子,Inc。
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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
Q1 Health Professions Pub Date : 2016-10-10 DOI: 10.1002/cpcy.9
Jan Vrána, Petr Cápal, Hana Šimková, Miroslava Karafiátová, Jana Čížková, Jaroslav Doležel

Analysis and sorting of plant chromosomes (plant flow cytogenetics) is a special application of flow cytometry in plant genomics and its success depends critically on sample quality. This unit describes the methodology in a stepwise manner, starting with the induction of cell cycle synchrony and accumulation of dividing cells in mitotic metaphase, and continues with the preparation of suspensions of intact mitotic chromosomes, flow analysis and sorting of chromosomes, and finally processing of the sorted chromosomes. Each step of the protocol is described in detail as some procedures have not been used widely. Supporting histograms are presented as well as hints on dealing with plant material; the utility of sorted chromosomes for plant genomics is also discussed. © 2016 by John Wiley & Sons, Inc.

植物染色体的分析和分选(植物流式细胞遗传学)是流式细胞术在植物基因组学中的一个特殊应用,其成功与否关键取决于样品质量。本单元以逐步的方式描述方法,从诱导细胞周期同步性和有丝分裂中期分裂细胞的积累开始,继续制备完整的有丝分裂染色体悬液,染色体的流动分析和分选,最后处理分选的染色体。由于有些程序尚未广泛使用,因此对协议的每个步骤都进行了详细描述。提供了支持直方图以及处理植物材料的提示;还讨论了染色体分类在植物基因组学中的应用。©2016 by John Wiley &儿子,Inc。
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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
Q1 Health Professions Pub Date : 2016-10-10 DOI: 10.1002/cpcy.8
Anja J. Gerrits, Andrew L. Frelinger III, Alan D. Michelson

In inflammatory and thrombotic syndromes, platelets aggregate with circulating leukocytes, especially monocytes and neutrophils. This leukocyte-platelet aggregate formation is initiated primarily through platelet surface expression of P-selectin (CD62P), following activation-dependent degranulation of α-granules, binding to its constitutively expressed counter-receptor, P-selectin glycoprotein ligand 1 (PSGL-1), on leukocytes. Monocyte-platelet aggregates are a more sensitive marker of platelet activation than platelet surface P-selectin. Detection of leukocyte-platelet aggregates is relatively simple by whole-blood flow cytometry. Light scatter and at least one leukocyte-specific antibody are used to gate the desired population, and the presence of associated platelets is detected by immunostaining for abundant platelet-specific markers. © 2016 by John Wiley & Sons, Inc.

在炎症和血栓综合征中,血小板与循环白细胞聚集,尤其是单核细胞和中性粒细胞。白细胞-血小板聚集的形成主要是通过血小板表面p -选择素(CD62P)的表达开始的,随后α-颗粒的激活依赖性脱粒,结合其组成性表达的对抗受体,p -选择素糖蛋白配体1 (PSGL-1),在白细胞上。单核细胞血小板聚集比血小板表面p选择素更敏感的血小板活化标志物。全血流式细胞术检测白细胞-血小板聚集物相对简单。光散射和至少一种白细胞特异性抗体用于筛选所需的人群,并通过丰富的血小板特异性标记物的免疫染色检测相关血小板的存在。©2016 by John Wiley &儿子,Inc。
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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
Q1 Health Professions Pub Date : 2016-07-01 DOI: 10.1002/cpcy.1
William Telford, Karen Tamul, Jolene Bradford

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.

细胞凋亡是细胞生物学中的一个重要机制,在几乎所有器官系统中都起着重要的调节作用。在免疫系统中,它的作用范围从未成熟免疫细胞的发育和选择到成熟免疫反应的下调。细胞凋亡也是抗癌药物的主要作用机制。25年来,流式细胞术一直是分析悬浮细胞凋亡的首选方法。从最早的信号转导事件到细胞形状和粒度、蛋白质水解和染色质凝聚等后期形态学变化,已经设计了许多测定方法来测量凋亡过程中最早和最新的步骤。当结合多色检测同时确定几种凋亡特征时,这些检测尤其有效。流式细胞术的多参数特性使该技术特别适合于测量细胞凋亡。在本单元中,我们将介绍分析凋亡细胞中caspase活性的四种主要技术,结合膜联蛋白V和细胞通透性分析。这些相对简单的多参数分析是评估细胞死亡的有力技术。©2016 by John Wiley &儿子,Inc。
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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
Q1 Health Professions Pub Date : 2016-07-01 DOI: 10.1002/cpcy.6
Aaron B. Kantor, Wayne A. Moore, Stephen Meehan, David R. Parks

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.

我们提出了一种定量的方法来比较抗体染料试剂的亮度和估计每个细胞结合的抗体。该方法基于测试试剂和填充试剂与抗体捕获微球的互补结合。几个等分的抗体捕获珠被不同数量的测试偶联物染色。然后用含有不同荧光团的第二种共轭物填充珠上剩余的结合位点。最后,与填充共轭物相比,测试共轭物的荧光用于测量测试共轭物的相对亮度。test-fill方法的基本假设是,如果需要一个测试抗体的X分子来降低Y个单位的填充信号,则需要任何其他测试抗体的相同X分子来产生相同的效果。我们应用二次拟合来评估不同数量的测试试剂的测试填充信号关系。如果拟合接近线性,我们认为该测试试剂适合用于抗体结合的定量评价。为了校准每个抗体珠结合的抗体,使用每个抗体1个PE分子的PE偶联物作为测试试剂,荧光刻度用Quantibrite PE珠校准。当每个抗体分子的荧光被确定为特定的偶联物时,该偶联物可用于测量每个细胞结合的抗体。这提供了不同的共轭亮度的比较,当进行仪器的统计光电子(Spe)尺度是已知的。©2016 by John Wiley &儿子,Inc。
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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
Q1 Health Professions 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

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.

高含量分析(High-content analysis, HCA)通过对相关信息内容的自动提取、多参数分析和分类,将原始光学显微镜图像转换为定量数据。与自动化高通量图像采集相结合,应用于化学物质或rnai试剂筛选的HCA被称为高含量筛选(HCS)。它在定量细胞表型方面的能力使HCA也适用于常规显微镜。然而,开发有效的HCA和生物信息学分析管道来获取HCS中具有生物学意义的数据是具有挑战性的。在这里,逐步发展的HCA测定方案和HCS生物信息学分析管道被描述。使用已发表的RNAi筛选的主要数据,该协议的功能通过应用于病灶粘附(FA)检测,多个FA特征的定量分析以及调节FA大小的信号通路的功能注释得到了证明。该分析和潜在策略的目标是研究人员在小规模或大型HCS实验中对亚细胞特征进行基于显微镜的定量分析。©2016 by John Wiley &儿子,Inc。
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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
Q1 Health Professions Pub Date : 2016-04-01 DOI: 10.1002/0471142956.cy0746s76
A. L. Henning, J. N. B. Sampson, B. McFarlin
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.
仪器设计和试剂开发的最新进展使流式细胞术领域的可用测量技术迅速发展。特别是,基于图像的流式细胞术扩展了传统流式细胞术的分析能力。直到最近,还不可能通过流式细胞术测量细胞内特定表型细胞的mRNA。在本方案中,描述了一种在体外暴露于修饰LDL的外周血单核细胞中完成PPAR γ mRNA和蛋白质的细胞内同步测量的方法。摄取修饰LDL后的PPAR - γ激活过程被认为在动脉粥样硬化的发展中起作用。使用扩增的FISH技术(PrimeFlow RNA Assay)可以检测低丰度的细胞内mRNA表达,从而可以测量PPAR - γ mRNA。该方案代表了作者实验室建立和验证新技术以评估免疫系统在慢性疾病中的作用的持续努力。©2016 by John Wiley & Sons, Inc。
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引用次数: 13
期刊
Current Protocols in Cytometry
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