Current Protocols in Cell Biology最新文献_第7页

Hepatitis C Virus Infection of Cultured Cells and Primary Human Hepatocytes 培养细胞和原代人肝细胞的丙型肝炎病毒感染

Current Protocols in Cell Biology Pub Date : 2018-07-30 DOI: 10.1002/cpcb.54

Brianna Lowey, Qisheng Li

{"title":"Hepatitis C Virus Infection of Cultured Cells and Primary Human Hepatocytes","authors":"Brianna Lowey, Qisheng Li","doi":"10.1002/cpcb.54","DOIUrl":"10.1002/cpcb.54","url":null,"abstract":"Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus in the family Flaviviridae with specific hepatotropism. HCV poses a significant health burden worldwide, frequently causing chronic infections associated with progressive liver damage and various extrahepatic manifestations. In recent years, the development of several permissive cell culture (HCVcc) systems has allowed for in vitro propagation of HCV, study of the viral life cycle and virus–host interactions, and identification of novel antivirals. Here we describe the use of human hepatoma cell lines Huh7 and HepG2/CD81/miR-122, as well as primary human hepatocytes (PHHs), for HCV infection and propagation. We also provide protocols for the quantitative analysis of intracellular and extracellular HCV RNA and detection of HCV core protein expression by immunostaining. In addition, we describe methods to manipulate cellular gene expression, including transfection of small interfering RNAs (siRNAs) targeting HCV host factors or overexpressing cellular microRNAs in hepatocytes, to assess their effects on productive HCV infection and liver pathogenesis. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.54","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36354049","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: 1

Isolation of Human Placental Mast Cells 人胎盘肥大细胞的分离

Current Protocols in Cell Biology Pub Date : 2018-07-25 DOI: 10.1002/cpcb.52

Soraya Mezouar, Amira Ben Amara, Joana Vitte, Jean-Louis Mege

引用次数: 9

Single-Cell, Time-Lapse Reactive Oxygen Species Detection in E. coli 大肠杆菌单细胞、延时活性氧检测

Current Protocols in Cell Biology Pub Date : 2018-07-20 DOI: 10.1002/cpcb.60

Zhilin Yang, Heejun Choi

引用次数: 8

Proteoglycan Isolation and Analysis 蛋白多糖的分离与分析

Current Protocols in Cell Biology Pub Date : 2018-06-21 DOI: 10.1002/cpcb.59

Anne Woods, John R. Couchman

引用次数: 3

Using Acutely Dissociated and Purified Oligodendrocyte Precursor Cells for High-Throughput Drug Screening to Identify Compounds that Promote Oligodendrocyte Differentiation 利用急性分离和纯化的少突胶质前体细胞进行高通量药物筛选以鉴定促进少突胶质细胞分化的化合物

Current Protocols in Cell Biology Pub Date : 2018-06-19 DOI: 10.1002/cpcb.49

Karen Lariosa-Willingham, Dmitri Leonoudakis

{"title":"Using Acutely Dissociated and Purified Oligodendrocyte Precursor Cells for High-Throughput Drug Screening to Identify Compounds that Promote Oligodendrocyte Differentiation","authors":"Karen Lariosa-Willingham, Dmitri Leonoudakis","doi":"10.1002/cpcb.49","DOIUrl":"10.1002/cpcb.49","url":null,"abstract":"Multiple sclerosis (MS) is an autoimmune disease that involves an immune-mediated inflammatory response in the central nervous system and optic nerve resulting in demyelination and neural degeneration, the cause of which is unknown. The adult central nervous system has the capacity to remyelinate axons by generating new oligodendrocytes (OLs). To identify clinical candidate compounds that may promote remyelination, we have developed a high-throughput screening (HTS) assay to identify compounds that promote the differentiation of oligodendrocyte precursor cells (OPCs) into OLs. Using acutely dissociated and purified rat OPCs coupled with immunofluorescent image quantification, we have developed an OL differentiation assay. Building on OPC culturing techniques developed over the past 30 years, we have scaled up the isolation and purification process to generate sufficient quantities for HTS. We then describe the use of these acutely derived OPCs in an assay designed to identify compounds that promote differentiation into OLs. We have validated this assay with a known promoter of differentiation, thyroid hormone, and subsequently used the assay to screen the NIH clinical collection library (Lariosa-Willingham, et al., 2016). © 2018 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"79 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.49","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36242124","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

Issue Information TOC 发布信息TOC

Current Protocols in Cell Biology Pub Date : 2018-06-01 DOI: 10.1002/nur.21824

引用次数: 0

Reconstitution of Mitotic Chromatids In Vitro 有丝分裂染色单体的体外重组

Current Protocols in Cell Biology Pub Date : 2018-05-24 DOI: 10.1002/cpcb.48

Keishi Shintomi, Tatsuya Hirano

引用次数: 3

Monitoring the Effects of Pharmacological Reagents on Mitochondrial Morphology 药理试剂对线粒体形态影响的监测

Current Protocols in Cell Biology Pub Date : 2018-05-24 DOI: 10.1002/cpcb.45

Dong Fu, Jennifer Lippincott-Schwartz

引用次数: 14

Transcription Factor–Mediated Differentiation of Human iPSCs into Neurons 转录因子介导的人多能干细胞向神经元的分化

Current Protocols in Cell Biology Pub Date : 2018-05-18 DOI: 10.1002/cpcb.51

Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward

{"title":"Transcription Factor–Mediated Differentiation of Human iPSCs into Neurons","authors":"Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward","doi":"10.1002/cpcb.51","DOIUrl":"10.1002/cpcb.51","url":null,"abstract":"Accurate modeling of human neuronal cell biology has been a long-standing challenge. However, methods to differentiate human induced pluripotent stem cells (iPSCs) to neurons have recently provided experimentally tractable cell models. Numerous methods that use small molecules to direct iPSCs into neuronal lineages have arisen in recent years. Unfortunately, these methods entail numerous challenges, including poor efficiency, variable cell type heterogeneity, and lengthy, expensive differentiation procedures. We recently developed a new method to generate stable transgenic lines of human iPSCs with doxycycline-inducible transcription factors at safe-harbor loci. Using a simple two-step protocol, these lines can be inducibly differentiated into either cortical (i3Neurons) or lower motor neurons (i3LMN) in a rapid, efficient, and scalable manner (Wang et al., 2017). In this manuscript, we describe a set of protocols to assist investigators in the culture and genetic engineering of iPSC lines to enable transcription factor–mediated differentiation of iPSCs into i3Neurons or i3LMNs, and we present neuronal culture conditions for various experimental applications. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"79 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-05-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.51","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36242125","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: 164

Isolation and Enrichment of Bacteriophages by Membrane Filtration Immobilization Technique 膜过滤固定化技术分离和富集噬菌体

Current Protocols in Cell Biology Pub Date : 2018-05-18 DOI: 10.1002/cpcb.41

Gaurav S. Ghugare, Vijay D. Nimkande, Krishna Khairnar

{"title":"Isolation and Enrichment of Bacteriophages by Membrane Filtration Immobilization Technique","authors":"Gaurav S. Ghugare, Vijay D. Nimkande, Krishna Khairnar","doi":"10.1002/cpcb.41","DOIUrl":"10.1002/cpcb.41","url":null,"abstract":"The method described here enables rapid bacteriophage isolation and enrichment of host-specific bacteriophages from an environmental sample. This is achieved by using a simple 0.45-µm Millipore membrane where a specific host is immobilized on the membrane and a sample suspected of containing bacteriophages is exposed to the immobilized cells with the help of a membrane filtration unit. This filtration step facilitates host-specific interaction of bacteriophages with the host and maximization of this interaction using a classic membrane filtration method. Under the effect of vacuum from a vacuum pump, a filter assembly provides a chance for every bacteriophage in the sample to interact with the specific host on the membrane filter. Our technique allows retaining specific bacteriophages on the membrane along with its host cells via adsorption; these adsorbed bacteriophages (along with their hosts) on a filter disc are then enriched in regular nutritive broth, tryptone soya broth (TSB), by incubation. With help of a plaque assay method, host-specific phages of various bacterial species can be isolated, segregated, and enriched. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"79 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-05-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.41","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36240193","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: 1