Current Protocols in Cytometry最新文献_第3页

Simultaneous Polychromatic Immunofluorescent Staining of Tissue Sections and Consecutive Imaging of up to Seven Parameters by Standard Confocal Microscopy 同时多色免疫荧光染色组织切片和连续成像多达七个参数的标准共聚焦显微镜

Current Protocols in Cytometry Pub Date : 2019-09-24 DOI: 10.1002/cpcy.64

Alfonso J. Schmidt, Johannes U. Mayer, Paul K. Wallace, Franca Ronchese, Kylie M. Price

{"title":"Simultaneous Polychromatic Immunofluorescent Staining of Tissue Sections and Consecutive Imaging of up to Seven Parameters by Standard Confocal Microscopy","authors":"Alfonso J. Schmidt, Johannes U. Mayer, Paul K. Wallace, Franca Ronchese, Kylie M. Price","doi":"10.1002/cpcy.64","DOIUrl":"10.1002/cpcy.64","url":null,"abstract":"Confocal microscopy has been an important imaging tool for life scientists for over 20 years. Early techniques focused on indirect staining processes that involved staining with an unconjugated primary antibody, followed by incubation with a secondary fluorescent antibody that would reveal and amplify the signal of the primary antibody. With more and more directly conjugated fluorescent primary antibodies becoming commercially available, staining with multiple fluorescent primary antibodies is now more frequent. To date, staining with up to three primary antibodies and a nuclear dye is widely practiced.Here, we describe an important improvement to the standard polychromatic immunofluorescent staining protocol that allows the simultaneous detection of seven fluorescent parameters using a standard confocal laser scanning microscope with four laser lines and four photomultiplier tubes. By incorporating recently available tandem dyes that emit in the blue and violet regions of the visible light spectrum (Brilliant Blue and Brilliant Violet), we were able to differentiate several additional fluorochromes simultaneously. Due to the added complexity of 7-color immunofluorescent imaging, we developed a clear methodology to optimize antibody concentrations and simple guidelines on how to identify and correct non-specific signals. These are detailed in the following protocol. © 2019 by John Wiley & Sons, Inc.Basic Protocol: 7-Color immunofluorescent staining protocol using directly conjugated antibodiesSupport Protocol 1: Antibody titration protocolSupport Protocol 2: Spillover optimization protocol","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"91 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.64","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46417069","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: 8

Enumeration of Fetal Red Blood Cells, Hemoglobin-Specific RBC Cells, and F Reticulocytes in Human Blood. 人血液中胎儿红细胞、血红蛋白特异性红细胞和网织红细胞的计数。

Current Protocols in Cytometry Pub Date : 2019-09-01 DOI: 10.1002/cpcy.56

Bruce H Davis

{"title":"Enumeration of Fetal Red Blood Cells, Hemoglobin-Specific RBC Cells, and F Reticulocytes in Human Blood.","authors":"Bruce H Davis","doi":"10.1002/cpcy.56","DOIUrl":"https://doi.org/10.1002/cpcy.56","url":null,"abstract":"Recent advances in analytical cytometry have improved diagnostic tools for the study of erythropoiesis in anemic patients and resolution of differential diagnosis in diseases of the erythron. This article presents three applications of red blood cell (RBC) analysis-quantitation of fetal red cells, F-cell enumeration, and F-reticulocyte analysis-which improve diagnostic precision, sensitivity, and specificity, and provide better laboratory indicators of therapeutic efficacy in a variety of hematologic and obstetric disorders. Such advances also include the measurement and quantitation of RBC hemoglobins and their relative ribonucleic acid levels. These advances not only promise to improve diagnostic accuracy and laboratory precision over techniques such as the traditional manual reticulocyte counting method and the Kleihauer-Betke stain method for evaluating fetomaternal hemorrhage (FMH), but also serve as tools for newer assays of anemia diagnosis and improved clinical outcomes. In addition to the primary methods, supporting techniques for preparing spiked controls, automating data analysis, setting up a fetal hemoglobin acquisition protocol, and assaying reticulocytes using thiazole orange are also presented. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"90 1","pages":"e56"},"PeriodicalIF":0.0,"publicationDate":"2019-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.56","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37511156","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

Cell Volume Measurements by Optical Transmission Microscopy. 用光学透射显微镜测量细胞体积。

Current Protocols in Cytometry Pub Date : 2019-09-01 DOI: 10.1002/cpcy.62

Michael A Model

引用次数: 0

Monitoring of Measurable Residual Disease in Multiple Myeloma by Multiparametric Flow Cytometry 多参数流式细胞术监测多发性骨髓瘤可测残余疾病

Current Protocols in Cytometry Pub Date : 2019-09-01 DOI: 10.1002/cpcy.63

K. Soh, P. Wallace

{"title":"Monitoring of Measurable Residual Disease in Multiple Myeloma by Multiparametric Flow Cytometry","authors":"K. Soh, P. Wallace","doi":"10.1002/cpcy.63","DOIUrl":"https://doi.org/10.1002/cpcy.63","url":null,"abstract":"Recent interest in high‐sensitivity multiple myeloma (MM) measurable residual disease (MRD) testing is a direct consequence of the high‐quality responses achieved using novel therapeutic agents and better treatment strategies. Traditional diagnostic measures such as immunohistochemistry and morphology have detection sensitivities of only 10−2 to 10−3, which do not reliably predict progression‐free survival (PFS) or overall survival (OS) after these treatments. Contemporary monitoring of MM MRD has switched to more sensitive platforms such as allele‐specific oligonucleotide quantitative polymerase chain reaction (ASO‐qPCR), next‐generation sequencing (NGS), and multiparametric flow cytometry (MFC). Though both ASO‐qPCR and NGS have excellent detection sensitivities (10−5 to 10−6), both technologies have lower applicability when compared to MFC. Conventional MFC can easily reach a detection sensitivity of 10−4 and when optimized can achieve a sensitivity of 10−5 to 10−6. Current consensus guidelines require a minimum of 2 million and recommend 5 million events be acquired to reach a minimum sensitivity of 10−5. As conventional immunophenotyping protocols are unable to attain these numbers, alternative MFC staining procedures are required. This article describes two high‐sensitivity MFC approaches that can be used for MM MRD testing. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.63","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43567538","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: 15

Issue Information TOC 发布信息TOC

Current Protocols in Cytometry Pub Date : 2019-09-01 DOI: 10.1002/cpcy.49

引用次数: 0

Issue Information TOC 发布信息TOC

Current Protocols in Cytometry Pub Date : 2019-06-20 DOI: 10.1002/cpcy.48

引用次数: 0

Detection of Histone H2AX Phosphorylation on Ser-139 as an Indicator of DNA Damage Ser-139上组蛋白H2AX磷酸化作为DNA损伤指标的检测

Current Protocols in Cytometry Pub Date : 2019-04-22 DOI: 10.1002/cpcy.55

Hong Zhao, Xuan Huang, H. Dorota Halicka, Zbigniew Darzynkiewicz

引用次数: 13

Issue Information TOC 发布信息TOC

Current Protocols in Cytometry Pub Date : 2019-03-18 DOI: 10.1002/cpcy.47

{"title":"Issue Information TOC","authors":"","doi":"10.1002/cpcy.47","DOIUrl":"10.1002/cpcy.47","url":null,"abstract":"Cover: In Eller et al. (https://doi.org/10.1002/cpcy.54), Hierarchical gates to evaluate fluorescent cells. Gates are applied identically to all samples. The P1 gate outlines the uniform population of HeLa cells for evaluation (typically >75% of total events). Under these specific conditions, events in the red region represent cellular debris or mechanically disrupted cells; if >50% of events are in this region it indicates an overall unhealthy sample. From the P1/cell population, single cells are identified by the P2 gate using their size distribution (typically >90% of P1). The P3/fluorescent population is derived from the P2/single cell population. The P3 fluorescent cell population is defined by a gate that excludes non-fluorescent cells in the untransfected sample (gray) and includes transfected cells (green, typically >50% of P2). There should be 10,000 events in P3 for an accurate evaluation. To calibrate the sensor in cells, an additional gate is required to identify the permeabilized and equilibrated cells. This P4 gate (typically >85% of P3) is derived from uniform, single cells in P2, and is delineated by equilibrated intracellular propidium iodide (PI). Fluorescence is then evaluated in P5, which is derived from P4. In this example, a gray density contour plot in the lower left quadrant represents cells that do no express either sensor or cpVenus. Cells that express either sensor or cpVenus populate P5, and the fluorescence of the populations treated with either equilibrated buffer (red) or 500 μM NAD+ (indigo) are overlaid. Data was collected on a BD Fortessa flow cytometer and analyzed on FlowJo V10 software. See e54.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture>\u0000 </div>\u0000 </figure>","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"88 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.47","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44135487","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

Issue Information TOC 发布信息TOC

Current Protocols in Cytometry Pub Date : 2019-01-02 DOI: 10.1002/cpcy.46

引用次数: 0

Flow Cytometry Analysis of Free Intracellular NAD+ Using a Targeted Biosensor 使用靶向生物传感器的细胞内游离NAD+的流式细胞术分析

Current Protocols in Cytometry Pub Date : 2018-12-17 DOI: 10.1002/cpcy.54

Jared M. Eller, Melissa L. Stewart, Alexandria J. Slepian, Sheila Markwardt, Jack Wiedrick, Michael S. Cohen, Richard H. Goodman, Xiaolu A. Cambronne

{"title":"Flow Cytometry Analysis of Free Intracellular NAD+ Using a Targeted Biosensor","authors":"Jared M. Eller, Melissa L. Stewart, Alexandria J. Slepian, Sheila Markwardt, Jack Wiedrick, Michael S. Cohen, Richard H. Goodman, Xiaolu A. Cambronne","doi":"10.1002/cpcy.54","DOIUrl":"10.1002/cpcy.54","url":null,"abstract":"Flow cytometry approaches combined with a genetically encoded targeted fluorescent biosensor are used to determine the subcellular compartmental availability of the oxidized form of nicotinamide adenine dinucleotide (NAD+). The availability of free NAD+ can affect the activities of NAD+-consuming enzymes such as sirtuin, PARP/ARTD, and cyclic ADPR-hydrolase family members. Many methods for measuring the NAD+ available to these enzymes are limited because they cannot determine free NAD+ as it exists in various subcellular compartments distinctly from bound NAD+ or NADH. Here, an approach to express the sensor in mammalian cells, monitor NAD+-dependent fluorescence intensity changes using flow cytometry approaches, and analyze data obtained is described. The benefit of flow cytometry approaches with the NAD+ sensor is the ability to monitor compartmentalized free NAD+ fluctuations simultaneously within many cells, which greatly facilitates analyses and calibration. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"88 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.54","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36777429","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: 5