Current Protocols in Cell Biology最新文献_第5页

Visualizing Secretory Cargo Transport in Budding Yeast 出芽酵母分泌货物运输的可视化

Current Protocols in Cell Biology Pub Date : 2018-11-10 DOI: 10.1002/cpcb.80

Jason C. Casler, Benjamin S. Glick

引用次数: 8

Single Molecule RNA FISH (smFISH) in Whole-Mount Mouse Embryonic Organs 全贴装小鼠胚胎器官中的单分子RNA FISH (smFISH)

Current Protocols in Cell Biology Pub Date : 2018-11-05 DOI: 10.1002/cpcb.79

Shaohe Wang

引用次数: 14

Generating Embryonic Salivary Gland Organoids 产生胚胎唾液腺类器官

Current Protocols in Cell Biology Pub Date : 2018-11-05 DOI: 10.1002/cpcb.76

Zeinab F. Hosseini, Deirdre A. Nelson, Nicholas Moskwa, Melinda Larsen

引用次数: 11

Visualization of Genomic Loci in Living Cells with BiFC-TALE 利用bbc - tale可视化活细胞基因组位点

Current Protocols in Cell Biology Pub Date : 2018-10-30 DOI: 10.1002/cpcb.78

Huan Hu, Xiaojing Yang, Chao Tang

引用次数: 1

Studying Glycolytic Oscillations in Individual Yeast Cells by Combining Fluorescence Microscopy with Microfluidics and Optical Tweezers 结合荧光显微镜、微流体和光学镊子研究单个酵母细胞中的糖酵解振荡

Current Protocols in Cell Biology Pub Date : 2018-10-17 DOI: 10.1002/cpcb.70

Anna-Karin Gustavsson, Amin A. Banaeiyan, David D. van Niekerk, Jacky L. Snoep, Caroline B. Adiels, Mattias Goksör

{"title":"Studying Glycolytic Oscillations in Individual Yeast Cells by Combining Fluorescence Microscopy with Microfluidics and Optical Tweezers","authors":"Anna-Karin Gustavsson, Amin A. Banaeiyan, David D. van Niekerk, Jacky L. Snoep, Caroline B. Adiels, Mattias Goksör","doi":"10.1002/cpcb.70","DOIUrl":"10.1002/cpcb.70","url":null,"abstract":"In this unit, we provide a clear exposition of the methodology employed to study dynamic responses in individual cells, using microfluidics for controlling and adjusting the cell environment, optical tweezers for precise cell positioning, and fluorescence microscopy for detecting intracellular responses. This unit focuses on the induction and study of glycolytic oscillations in single yeast cells, but the methodology can easily be adjusted to examine other biological questions and cell types. We present a step-by-step guide for fabrication of the microfluidic device, for alignment of the optical tweezers, for cell preparation, and for time-lapse imaging of glycolytic oscillations in single cells, including a discussion of common pitfalls. A user who follows the protocols should be able to detect clear metabolite time traces over the course of up to an hour that are indicative of dynamics on the second scale in individual cells during fast and reversible environmental adjustments. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"82 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.70","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36582424","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: 2

Analyzing Integrin-Dependent Adhesion 分析整合素依赖性粘附

Current Protocols in Cell Biology Pub Date : 2018-10-01 DOI: 10.1002/cpcb.69

A. Paul Mould

引用次数: 7

Identification, Localization, and Quantification of HIV Reservoirs Using Microscopy 鉴定，定位和定量艾滋病毒库使用显微镜

Current Protocols in Cell Biology Pub Date : 2018-09-28 DOI: 10.1002/cpcb.64

Lisa Prevedel, Nancy Ruel, Paul Castellano, Carla Smith, Shaily Malik, Courtney Villeux, Morgane Bomsel, Susan Morgello, Eliseo A. Eugenin

{"title":"Identification, Localization, and Quantification of HIV Reservoirs Using Microscopy","authors":"Lisa Prevedel, Nancy Ruel, Paul Castellano, Carla Smith, Shaily Malik, Courtney Villeux, Morgane Bomsel, Susan Morgello, Eliseo A. Eugenin","doi":"10.1002/cpcb.64","DOIUrl":"10.1002/cpcb.64","url":null,"abstract":"The major barrier to eradicating human immunodeficiency virus-1 (HIV) infection is the generation and extended survival of HIV reservoirs. In order to eradicate HIV infection, it is essential to detect, quantify, and characterize circulating and tissue-associated viral reservoirs in infected individuals. Currently, PCR-based technologies and Quantitative Viral Outgrowth Assays (Q-VOA) are the gold standards to detect viral reservoirs. However, these methods are limited to detecting circulating viral reservoirs, and it has been shown that they misrepresent the size of the reservoirs, largely because they detect only one component of the HIV life cycle and are unable to detect viral reservoirs in tissues. Here, we described the use of multiple detection systems to identify integrated HIV DNA or viral mRNA and several HIV proteins in circulating and tissue reservoirs using improved staining and microscopy techniques. We believe that this imaging-based approach for detecting HIV reservoirs will lead to breakthroughs necessary to eradicate these reservoirs. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"82 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.64","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36529589","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: 20

Fluorescence Activated Cell Sorting (FACS) in Genome-Wide Genetic Screening of Membrane Trafficking 荧光活化细胞分选(FACS)在膜运输全基因组遗传筛选中的应用

Current Protocols in Cell Biology Pub Date : 2018-09-28 DOI: 10.1002/cpcb.68

Bridget L. Menasche, Lauren Crisman, Daniel R. Gulbranson, Eric M. Davis, Haijia Yu, Jingshi Shen

{"title":"Fluorescence Activated Cell Sorting (FACS) in Genome-Wide Genetic Screening of Membrane Trafficking","authors":"Bridget L. Menasche, Lauren Crisman, Daniel R. Gulbranson, Eric M. Davis, Haijia Yu, Jingshi Shen","doi":"10.1002/cpcb.68","DOIUrl":"10.1002/cpcb.68","url":null,"abstract":"About one-third of cellular proteins in eukaryotic cells are localized to membrane-enclosed organelles in the endomembrane system. Trafficking of these membrane proteins (including soluble lumenal proteins) among the organelles is mediated by small sac-like vesicles. Vesicle-mediated membrane trafficking regulates a broad range of biological processes, many of which are still poorly understood at the molecular level. A powerful approach to dissect a vesicle-mediated membrane trafficking pathway is unbiased genome-wide genetic screening, which only recently became possible in mammalian cells with the isolation of haploid human cell lines and the development of CRISPR-Cas9 genome editing. Here, we describe a FACS-based method to select populations of live mutant cells based on the surface levels of endogenous proteins or engineered reporters. Collection of these mutant populations enables subsequent deep sequencing and bioinformatics analysis to identify genes that regulate the trafficking pathway. This method can be readily adapted to genetically dissect a broad range of mammalian membrane trafficking processes using haploid genetics or CRISPR-Cas9 screens. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"82 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.68","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36533164","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

Isolation, Culture, and Differentiation of Mammary Epithelial Stem/Progenitor Cells from Fresh or Ex Vivo Cultured Human Breast Tissue 从新鲜或离体培养的人乳腺组织中分离、培养和分化乳腺上皮干/祖细胞

Current Protocols in Cell Biology Pub Date : 2018-09-28 DOI: 10.1002/cpcb.65

Guang Chen, Hakim Bouamar, Lu-Zhe Sun

{"title":"Isolation, Culture, and Differentiation of Mammary Epithelial Stem/Progenitor Cells from Fresh or Ex Vivo Cultured Human Breast Tissue","authors":"Guang Chen, Hakim Bouamar, Lu-Zhe Sun","doi":"10.1002/cpcb.65","DOIUrl":"10.1002/cpcb.65","url":null,"abstract":"In vivo transplantation is the gold standard method for characterization of stem/progenitor cell self-renewal, tissue regeneration, and tumorigenesis. The method requires an enriched population of stem cells that represent a small fraction of a given tissue. An enriched population of stem/progenitor cells increases the likelihood of engraftment and reduces the number of recipient animals needed for in vivo transplantation. Methods for mammosphere formation by mammary epithelial stem and progenitor cells have been widely adopted for enriching stem/progenitor cells, allowing researchers to study genetic and epigenetic properties, interaction with other cell types, and differentiation and oncogenic transformation. The generation of mammospheres is complex, however, involving many steps and requiring particular skill. Here we describe a detailed mammosphere protocol, including isolation and culture of human primary mammary epithelial stem/progenitor cells and their differentiation and passage in 3D organoid culture. We also describe a protocol for ex vivo culture of fresh human breast tissue for use in assays of clinical treatment. Step-by-step instructions detail tissue handling through passage of the stem/progenitor cell-generated 3D organoids, which can be used to assess the properties, function, and neoplastic transformation of mammary stem/progenitor cells. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"82 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.65","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36529590","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

Initiation, Expansion, and Cryopreservation of Human Primary Tissue-Derived Normal and Diseased Organoids in Embedded Three-Dimensional Culture 人原代组织衍生的正常和病变类器官在嵌入三维培养中的起始、扩增和低温保存

Current Protocols in Cell Biology Pub Date : 2018-09-28 DOI: 10.1002/cpcb.66

James Clinton, Penney McWilliams-Koeppen

引用次数: 24