Current Protocols in Cytometry最新文献_第2页

Practical Guidelines for Collection, Manipulation and Inactivation of SARS-CoV-2 and COVID-19 Clinical Specimens. SARS-CoV-2和COVID-19临床标本采集、操作和灭活实用指南

Current Protocols in Cytometry Pub Date : 2020-06-01 DOI: 10.1002/cpcy.77

William Bain, Janet S Lee, Alan M Watson, Molly S Stitt-Fischer

{"title":"Practical Guidelines for Collection, Manipulation and Inactivation of SARS-CoV-2 and COVID-19 Clinical Specimens.","authors":"William Bain, Janet S Lee, Alan M Watson, Molly S Stitt-Fischer","doi":"10.1002/cpcy.77","DOIUrl":"https://doi.org/10.1002/cpcy.77","url":null,"abstract":"SARS-CoV-2 is a novel coronavirus that causes the acute respiratory disease-Coronavirus disease 2019 (COVID-19)-which has led to a global health crisis. Currently, no prophylactics or therapies exist to control virus spread or mitigate the disease. Thus, the risk of infection for physicians and scientists is high, requiring work to be conducted in Biosafety Level-3 (BSL-3) facilities if virus will be isolated or propagated. However, inactivation of the virus can enable safe handling at a reduced biosafety level, making samples accessible to a diverse array of institutions and investigators. Institutions of all types have an immediate need for guidelines that outline safe collection, handling, and inactivation of samples suspected to contain active virus. Here we provide a practical guide for physicians and researchers wishing to work with materials from patients who are COVID-19 positive or suspected positive. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Practical guidelines for the safe collection and handling of specimens collected from COVID-19 and suspected COVID-19 patients Basic Protocol 2: Inactivating SARS-CoV-2.","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"93 1","pages":"e77"},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.77","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38013420","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: 43

Automated Motion Tracking and Data Extraction for Red Blood Cell Biomechanics. 红细胞生物力学的自动运动跟踪和数据提取。

Current Protocols in Cytometry Pub Date : 2020-06-01 DOI: 10.1002/cpcy.75

Arun Kumar, Brendan R Schmidt, Zyrina Alura C Sanchez, Feyza Yazar, Ronald W Davis, Anand K Ramasubramanian, Amit K Saha

{"title":"Automated Motion Tracking and Data Extraction for Red Blood Cell Biomechanics.","authors":"Arun Kumar, Brendan R Schmidt, Zyrina Alura C Sanchez, Feyza Yazar, Ronald W Davis, Anand K Ramasubramanian, Amit K Saha","doi":"10.1002/cpcy.75","DOIUrl":"https://doi.org/10.1002/cpcy.75","url":null,"abstract":"Red blood cell biomechanics can provide us with a deeper understanding of macroscopic physiology and have the potential of being used for diagnostic purposes. In diseases like sickle cell anemia and malaria, reduced red blood cell deformability can be used as a biomarker, leading to further assays and diagnoses. A microfluidic system is useful for studying these biomechanical properties. We can observe detailed red blood cell mechanical behavior as they flow through microcapillaries using high-speed imaging and microscopy. Microfluidic devices are advantageous over traditional methods because they can serve as high-throughput tests. However, to rapidly analyze thousands of cells, there is a need for powerful image processing tools and software automation. We describe a workflow process using Image-Pro to identify and track red blood cells in a video, take measurements, and export the data for use in statistical analysis tools. The information in this protocol can be applied to large-scale blood studies where entire cell populations need to be analyzed from many cohorts of donors. © 2020 The Authors. Basic Protocol 1: Enhancing raw video for motion tracking Basic Protocol 2: Extracting motion tracking data from enhanced video.","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"93 1","pages":"e75"},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.75","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37920431","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

Panel Design and Optimization for High-Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry 使用光谱流式细胞术的高维免疫表型分析的面板设计和优化

Current Protocols in Cytometry Pub Date : 2020-03-05 DOI: 10.1002/cpcy.70

Laura Ferrer-Font, Christophe Pellefigues, Johannes U. Mayer, Sam J. Small, Maria C. Jaimes, Kylie M. Price

{"title":"Panel Design and Optimization for High-Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry","authors":"Laura Ferrer-Font, Christophe Pellefigues, Johannes U. Mayer, Sam J. Small, Maria C. Jaimes, Kylie M. Price","doi":"10.1002/cpcy.70","DOIUrl":"10.1002/cpcy.70","url":null,"abstract":"Technological advances in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system have led to the development of large (20+ parameters) flow cytometry panels. However, as panel complexity and size increase, so does the difficulty involved in designing a high-quality panel, accessing the instrumentation capable of accommodating large numbers of parameters, and analyzing such high-dimensional data. A recent advancement is spectral flow cytometry, which in contrast to conventional flow cytometry distinguishes the full emission spectrum of each fluorophore across all lasers, rather than identifying only the peak of emission. Fluorophores with a similar emission maximum but distinct off-peak signatures can therefore be accommodated within the same flow cytometry panel, allowing greater flexibility in terms of panel design and fluorophore detection. Here, we highlight the specific characteristics of spectral flow cytometry and aim to guide users through the process of building, designing, and optimizing high-dimensional spectral flow cytometry panels using a comprehensive step-by-step protocol. Special considerations are also given for using highly overlapping dyes, and a logical selection process for optimal marker-fluorophore assignment is provided. © 2020 by John Wiley & Sons, Inc.","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"92 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.70","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37718930","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: 66

Training Novices in Generation and Analysis of High-Dimensional Human Cell Phospho-Flow Cytometry Data. 培训新手高维人细胞磷流式细胞术数据的生成和分析。

Current Protocols in Cytometry Pub Date : 2020-03-01 DOI: 10.1002/cpcy.71

Caroline E Roe, Madeline J Hayes, Sierra M Barone, Jonathan M Irish

{"title":"Training Novices in Generation and Analysis of High-Dimensional Human Cell Phospho-Flow Cytometry Data.","authors":"Caroline E Roe, Madeline J Hayes, Sierra M Barone, Jonathan M Irish","doi":"10.1002/cpcy.71","DOIUrl":"https://doi.org/10.1002/cpcy.71","url":null,"abstract":"This article presents a single experiment designed to introduce a trainee to multiple advanced bench and analysis techniques, including high-dimensional cytometry, profiling cell signaling networks, functional assays with primary human tissue, and single-cell analysis with machine learning tools. The trainee is expected to have only minimal laboratory experience and is not required to have any prior training in flow cytometry, immunology, or data science. This article aims to introduce the advanced research areas with a design that is robust enough that novice trainees will succeed, flexible enough to allow some project customization, and fundamental enough that the skills and knowledge gained will provide a template for future experiments. For advanced users, the updated phospho-flow protocol and the established controls, best practices, and expected outcomes presented here also provide a framework for adapting these tools in new areas with unexplored biology. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Phospho-protein stimulation and mass cytometry data collection Support Protocol: Analysis of signaling mass cytometry data.","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"93 1","pages":"e71"},"PeriodicalIF":0.0,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.71","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37803959","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: 7

Single-Cell Analysis of Cytokine mRNA and Protein Expression by Flow Cytometry 单细胞细胞因子mRNA和蛋白表达的流式细胞术分析

Current Protocols in Cytometry Pub Date : 2020-02-24 DOI: 10.1002/cpcy.69

Rubina Pal, Jayne Schaubhut, Darcey Clark, Lynette Brown, Jennifer J. Stewart

{"title":"Single-Cell Analysis of Cytokine mRNA and Protein Expression by Flow Cytometry","authors":"Rubina Pal, Jayne Schaubhut, Darcey Clark, Lynette Brown, Jennifer J. Stewart","doi":"10.1002/cpcy.69","DOIUrl":"10.1002/cpcy.69","url":null,"abstract":"Understanding how immune cells respond to external stimuli such as pathogens or drugs is a key component of biomedical research. Critical to the immune response are the expression of cell-surface receptors and the secretion of cytokines, which are tightly regulated by gene expression and protein synthesis. Previously, cytokine mRNA expression levels have been measured from bulk analysis of heterogeneous or sorted cell populations, and the correlation between cytokine mRNA expression and protein levels using these techniques can be highly variable. Flow cytometry is used to monitor changes in cell-surface and intracellular proteins, but some proteins such as cytokines may be transient and difficult to measure. Thus, a flow cytometry method that can simultaneously measure cytokine mRNA and protein levels in single cells is a very powerful tool. We defined a flow cytometry method that combines the conventional measurement of T cell surface proteins (CD45, CD3, CD4, CD8) and intracellular cytokines (IL-2, INF-γ) with fluorescent in situ hybridization and branched DNA technology for amplification and detection of IL-2 and INF-γ mRNA transcripts in activated T cells. This method has been applied to frozen peripheral mononuclear blood cells (PBMCs) and frozen blood samples, making it applicable to clinical trial specimens that require shipment to the test site. In CD4+ cells from activated PBMCs, the concordance between mRNA and protein levels was 41% for IL-2 and 21% for and INF-γ. In CD8+ cells from activated PBMCs, the concordance was 15% for IL-2 and 32% for INF-γ. © 2020 by John Wiley & Sons, Inc.Basic Protocol: Detection of IL-2 and IFN-γ mRNA and protein expression in frozen PBMCsAlternate Protocol: Detection of IL-2 and IFN-γ mRNA and protein expression in frozen blood","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"92 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.69","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37670992","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

Confocal Microscopy: Principles and Modern Practices 共聚焦显微镜:原理和现代实践

Current Protocols in Cytometry Pub Date : 2019-12-26 DOI: 10.1002/cpcy.68

Amicia D. Elliott

{"title":"Confocal Microscopy: Principles and Modern Practices","authors":"Amicia D. Elliott","doi":"10.1002/cpcy.68","DOIUrl":"10.1002/cpcy.68","url":null,"abstract":"In light microscopy, illuminating light is passed through the sample as uniformly as possible over the field of view. For thicker samples, where the objective lens does not have sufficient depth of focus, light from sample planes above and below the focal plane will also be detected. The out-of-focus light will add blur to the image, reducing the resolution. In fluorescence microscopy, any dye molecules in the field of view will be stimulated, including those in out-of-focus planes. Confocal microscopy provides a means of rejecting the out-of-focus light from the detector such that it does not contribute blur to the images being collected. This technique allows for high-resolution imaging in thick tissues.In a confocal microscope, the illumination and detection optics are focused on the same diffraction-limited spot in the sample, which is the only spot imaged by the detector during a confocal scan. To generate a complete image, the spot must be moved over the sample and data collected point by point. A significant advantage of the confocal microscope is the optical sectioning provided, which allows for 3D reconstruction of a sample from high-resolution stacks of images. Several types of confocal microscopes have been developed for this purpose, and each has different advantages and disadvantages. This article provides a concise introduction to confocal microscopy. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"92 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.68","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37491990","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: 93

Issue Information TOC 发布信息TOC

Current Protocols in Cytometry Pub Date : 2019-12-19 DOI: 10.1002/cpcy.57

引用次数: 0

Basics of Expansion Microscopy 扩展显微镜基础知识

Current Protocols in Cytometry Pub Date : 2019-10-24 DOI: 10.1002/cpcy.67

Aleksandra Klimas, Brendan Gallagher, Yongxin Zhao

引用次数: 2

Immunophenotypic Detection of Measurable Residual (Stem Cell) Disease Using LAIP Approach in Acute Myeloid Leukemia 应用LAIP方法检测急性髓系白血病可测量残余(干细胞)病变的免疫表型

Current Protocols in Cytometry Pub Date : 2019-10-23 DOI: 10.1002/cpcy.66

Wendelien Zeijlemaker, Angele Kelder, Jacqueline Cloos, Gerrit Jan Schuurhuis

{"title":"Immunophenotypic Detection of Measurable Residual (Stem Cell) Disease Using LAIP Approach in Acute Myeloid Leukemia","authors":"Wendelien Zeijlemaker, Angele Kelder, Jacqueline Cloos, Gerrit Jan Schuurhuis","doi":"10.1002/cpcy.66","DOIUrl":"10.1002/cpcy.66","url":null,"abstract":"Half of the patients with acute myeloid leukemia (AML), who achieve complete remission after chemotherapy treatment, will ultimately experience a relapse. Measurable residual disease (MRD) is an important post-treatment risk factor in AML, because it gives additional information about the depth of the remission. Within MRD, the small population of leukemic stem cells (LSCs) is thought to be at the base of the actual relapse. In this protocol, the flow cytometric detection of MRD and LSCs herein is outlined. We give a detailed overview of the sampling procedures for optimal multiparameter flow cytometry assessment of both MRD and LSC, using leukemia associated immunophenotypes (LAIPs) and LSC markers. Moreover, an overview of the gating strategies to detect LAIPs and LSC markers is provided. This protocol serves as guidance for flow cytometric detection of measurable residual (stem cell) disease necessary for proper therapeutic decision making in AML patients. © 2019 The Authors.Basic Protocol 1: Immunophenotypic LAIP detection for measurable residual disease monitoringBasic Protocol 2: Immunophenotypic detection of CD34+CD38− leukemic stem cells","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"91 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.66","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43842201","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: 25

Assessment of DNA Susceptibility to Denaturation as a Marker of Chromatin Structure 作为染色质结构标记的DNA变性易感性评估

Current Protocols in Cytometry Pub Date : 2019-10-16 DOI: 10.1002/cpcy.65

Zbigniew Darzynkiewicz, Dorota H. Halicka, Hong Zhao, Jiangwei Li

{"title":"Assessment of DNA Susceptibility to Denaturation as a Marker of Chromatin Structure","authors":"Zbigniew Darzynkiewicz, Dorota H. Halicka, Hong Zhao, Jiangwei Li","doi":"10.1002/cpcy.65","DOIUrl":"10.1002/cpcy.65","url":null,"abstract":"The susceptibility of DNA in situ to denaturation is modulated by its interactions with histone and nonhistone proteins, as well as with other chromatin components related to the maintenance of the 3D nuclear structure. Measurement of DNA proclivity to denature by cytometry provides insight into chromatin structure and thus can be used to recognize cells in different phases of the cell cycle, including mitosis, quiescence (G0), and apoptosis, as well as to identify the effects of drugs that modify chromatin structure. Particularly useful is the method's ability to detect chromatin changes in sperm cells related to DNA fragmentation and infertility. This article presents a flow cytometric procedure for assessing DNA denaturation based on application of the metachromatic property of acridine orange (AO) to differentially stain single- versus double-stranded DNA. This approach circumvents limitations of biochemical methods of examining DNA denaturation, in particular the fact that the latter destroy higher orders of chromatin structure and that, being applied to bulk cell populations, they cannot detect heterogeneity of individual cells. Because the metachromatic properties of AO have also found application in other cytometric procedures, such as differential staining of RNA versus DNA and assessment of lysosomal proton pump including autophagy, to avert confusion between these approaches and the use of this dye in the DNA denaturation assay, these AO applications are briefly outlined in this unit as well. © 2019 by John Wiley & Sons, Inc.Basic Protocol: Differential staining of single- versus double-stranded DNA with acridine orange","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"91 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.65","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42692178","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