Current Protocols in Cell Biology最新文献_第3页

Current Protocols in Cell Biology Pub Date : 2019-12-01 DOI: 10.1002/cpcb.75

引用次数: 0

Preparation and Co-Culture of iPSC-Derived Dopaminergic Neurons and Astrocytes 多能干细胞多巴胺能神经元与星形胶质细胞的制备与共培养

Current Protocols in Cell Biology Pub Date : 2019-10-04 DOI: 10.1002/cpcb.98

Aurelie de Rus Jacquet

{"title":"Preparation and Co-Culture of iPSC-Derived Dopaminergic Neurons and Astrocytes","authors":"Aurelie de Rus Jacquet","doi":"10.1002/cpcb.98","DOIUrl":"https://doi.org/10.1002/cpcb.98","url":null,"abstract":"Induced pluripotent stem cell (iPSC)-based models are powerful tools to study neurodegenerative diseases such as Parkinson's disease. The differentiation of patient-derived neurons and astrocytes allows investigation of the molecular mechanisms responsible for disease onset and development. In particular, these two cell types can be mono- or co-cultured to study the influence of cell-autonomous and non-cell-autonomous contributors to neurodegenerative diseases. We developed a streamlined procedure to produce high-quality/high-purity cultures of dopaminergic neurons and astrocytes that originate from the same population of midbrain floor-plate progenitors. This unit describes differentiation, quality control, culture parameters, and troubleshooting tips to ensure the highest quality and reproducibility of research results. © 2019 The Authors.Basic Protocol 1: Differentiation of iPSCs into midbrain-patterned neural progenitor cellsSupport Protocol: Quality control of neural progenitor cellsBasic Protocol 2: Differentiation of neural progenitor cells into astrocytesBasic Protocol 3: Differentiation of neural progenitor cells into dopaminergic neuronsBasic Protocol 4: Co-culture of iPSC-derived neurons and astrocytes","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"85 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.98","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91800999","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: 14

In Situ Visualization of Telomere Length, Telomere Elongation, and TERT Expression in Single Cells 单细胞中端粒长度、端粒伸长和TERT表达的原位可视化

Current Protocols in Cell Biology Pub Date : 2019-10-03 DOI: 10.1002/cpcb.97

Ajay Ravindranathan, Morgan E. Diolaiti, Beth A. Cimini, Bradley A. Stohr

{"title":"In Situ Visualization of Telomere Length, Telomere Elongation, and TERT Expression in Single Cells","authors":"Ajay Ravindranathan, Morgan E. Diolaiti, Beth A. Cimini, Bradley A. Stohr","doi":"10.1002/cpcb.97","DOIUrl":"https://doi.org/10.1002/cpcb.97","url":null,"abstract":"Telomerase plays a critical role in cancer and aging by adding hexa-nucleotide repeats to the ends of telomeres and extending the cellular proliferative lifespan. The very low level of telomerase expression in most cell populations and the difficulty of detecting telomere elongation in single cells have limited the study of telomerase expression and function in individual cells of a heterogeneous population. The method described in this article combines single-molecule detection (RNAscope) of telomerase reverse transcriptase (TERT) with our previously described TSQ1 assay for in situ monitoring of telomere extension, thereby enabling detection of TERT expression, telomere length, and telomere elongation in single cells and providing a unique approach for studying the factors that regulate telomere elongation by telomerase. © 2019 by John Wiley & Sons, Inc.Basic Protocol 1: TSQ1 lentivirus productionBasic Protocol 2: TSQ1 lentiviral infection and platingBasic Protocol 3: RNAscope analysisBasic Protocol 4: TSQ1 PNA-FISH detection","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"85 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.97","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91798007","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

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Current Protocols in Cell Biology Pub Date : 2019-09-03 DOI: 10.1002/cpcb.74

引用次数: 0

Protein-Protein Interaction Mapping by 2C-BioID 2C-BioID蛋白-蛋白相互作用图谱

Current Protocols in Cell Biology Pub Date : 2019-08-27 DOI: 10.1002/cpcb.96

Alexandre Chojnowski, Hendrikje Werner, Matthew Cook, Radoslaw M. Sobota, Brian Burke, Colin L. Stewart

{"title":"Protein-Protein Interaction Mapping by 2C-BioID","authors":"Alexandre Chojnowski, Hendrikje Werner, Matthew Cook, Radoslaw M. Sobota, Brian Burke, Colin L. Stewart","doi":"10.1002/cpcb.96","DOIUrl":"10.1002/cpcb.96","url":null,"abstract":"Protein-protein interactions (PPIs) add an essential layer of complexity to the information encoded by the genome. Modulation of such interactions is a key feature of most, if not all, cellular activities and allows cells to respond rapidly to both internal and external signals and stimuli. In this respect, the development of the BioID assay to interrogate PPIs within a cellular context represents an important adjunct to the range of tools currently at researchers' disposal. To address some of its current limitations, we devised 2C-BioID, in which biotin ligase and the protein of interest remain as separate entities until induced to associate. This is accomplished using the well-established FKBP-FRB dimerization system (based on the rapamycin-induced binding of FK506 binding protein and FKBP12-rapamycin binding domain.). The design of 2C-BioID ensures that biotin ligase association with the protein of interest occurs only after addition of the rapamycin analogue AP21967. As such, 2C-BioID alleviates potential targeting issues and improves the ability to exclude false positives, thereby refining the specificity of BioID-generated interactomes. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"84 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.96","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91104408","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

Nuclei Isolation Staining (NIS) Method for Imaging Chromatin-Associated Proteins in Difficult Cell Types 细胞核分离染色(NIS)方法在困难细胞类型中成像染色质相关蛋白

Current Protocols in Cell Biology Pub Date : 2019-08-13 DOI: 10.1002/cpcb.94

Amy E. Neely, Xiaomin Bao

{"title":"Nuclei Isolation Staining (NIS) Method for Imaging Chromatin-Associated Proteins in Difficult Cell Types","authors":"Amy E. Neely, Xiaomin Bao","doi":"10.1002/cpcb.94","DOIUrl":"10.1002/cpcb.94","url":null,"abstract":"Spatial distribution of chromatin-associated proteins provides invaluable information for understanding gene regulation. Conventional immunostaining is widely used for labeling chromatin-associated proteins in many cell types. However, for a subset of difficult cell types, such as differentiated human keratinocytes, achieving high-quality immunostaining for nuclear proteins remains challenging. To overcome this technical barrier, we developed the nuclei isolation staining (NIS) method. In brief, NIS involves rapid isolation of nuclei from live cells, followed by fixation and staining of the nuclei directly on coverslips for subsequent high-magnification imaging. By removing the cytoplasmic contents and staining just the nuclei, this NIS method drastically improves antibody labeling efficiency for chromatin-associated proteins. In this article, we describe the development and a step-by-step protocol of NIS, using differentiated human keratinocytes as an example. We also discuss other applications, based on the principle of this NIS method, for understanding cell-type and cell-state specific gene regulation. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"84 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.94","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84070373","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

A Neuron-Glia Co-culture System for Studying Intercellular Lipid Transport 研究细胞间脂质转运的神经元-胶质共培养系统

Current Protocols in Cell Biology Pub Date : 2019-08-09 DOI: 10.1002/cpcb.95

Maria S. Ioannou, Zhe Liu, Jennifer Lippincott-Schwartz

引用次数: 15

Pinpointing the Genomic Localizations of Chromatin-Associated Proteins: The Yesterday, Today, and Tomorrow of ChIP-seq 染色质相关蛋白的基因组定位:ChIP-seq的昨天，今天和明天

Current Protocols in Cell Biology Pub Date : 2019-06-03 DOI: 10.1002/cpcb.89

Sarah M. Lloyd, Xiaomin Bao

引用次数: 10

Western Blotting with Solutions containing Nanoliter Volumes of Antibody 抗体体积为纳升的Western Blotting

Current Protocols in Cell Biology Pub Date : 2019-05-13 DOI: 10.1002/cpcb.87

David A. Cruz Walma, Joshua W. Collins

{"title":"Western Blotting with Solutions containing Nanoliter Volumes of Antibody","authors":"David A. Cruz Walma, Joshua W. Collins","doi":"10.1002/cpcb.87","DOIUrl":"10.1002/cpcb.87","url":null,"abstract":"Whether screening small mammal serum during antibody production or attempting to preserve a stock of precious antibody, this protocol's western blotting method using aliquots containing nanoliter volumes of antibody will benefit researchers. Time-tested western blotting workflows allowing separation and analysis of proteins are routinely utilized in clinical and laboratory settings. The necessity for relatively large quantities of antibody is a major limitation to this universal tool. This article provides a step-by-step protocol for detecting proteins of interest with solutions containing nanoliter volumes of antibody without altering the preceding gel electrophoresis and transfer methods. Important considerations, frequently encountered problems, and means of optimizing reproducibility are discussed. Complementary diagrams, images, and videos are provided. The protocol is demonstrated using 0.3 nanoliters of anti-serum to detect fibronectin in a human foreskin fibroblast cell line. Finally, two support protocols detailing methods of extracting proteins from cultured cells are reported. Published 2019. This article is a US Government work and is in the public domain in the USA.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"84 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-05-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.87","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86748416","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

Issue Information TOC 发布信息TOC

Current Protocols in Cell Biology Pub Date : 2019-05-08 DOI: 10.1002/cpcb.73

引用次数: 0