CSH protocols最新文献_第5页

Cultures of cerebellar granule neurons. 小脑颗粒神经元的培养。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.prot5107

Parizad M Bilimoria, Azad Bonni

{"title":"Cultures of cerebellar granule neurons.","authors":"Parizad M Bilimoria, Azad Bonni","doi":"10.1101/pdb.prot5107","DOIUrl":"https://doi.org/10.1101/pdb.prot5107","url":null,"abstract":"INTRODUCTIONPrimary cultures of granule neurons from the post-natal rat cerebellum provide an excellent model system for molecular and cell biological studies of neuronal development and function. The cerebellar cortex, with its highly organized structure and few neuronal subtypes, offers a well-characterized neural circuitry. Many fundamental insights into the processes of neuronal apoptosis, migration, and differentiation in the mammalian central nervous system have come from investigating granule neurons in vitro. Granule neurons are the most abundant type of neurons in the brain. In addition to the sheer volume of granule neurons, the homogeneity of the population and the fact that they can be transfected with ease render them ideal for elucidating the molecular basis of neuronal development. This protocol for isolating granule neurons from post-natal rats is relatively straightforward and quick, making use of standard enzymatic and mechanical dissociation methods. In a serum-based medium containing an inhibitor of mitosis, cerebellar granule neurons can be maintained with high purity. Axons and dendrites can be clearly distinguished on the basis of morphology and markers. For even greater versatility, this protocol for culturing granule neurons can be combined with knockout or transgenic technologies, or used in cerebellar slice overlay assays.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2008-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.prot5107","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29701742","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: 67

The Demosponge Amphimedon queenslandica: Reconstructing the Ancestral Metazoan Genome and Deciphering the Origin of Animal Multicellularity. 昆士兰的蠕形海绵Amphimedon:重建祖先后生动物基因组和破译动物多细胞起源。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.emo108

Bernard M Degnan, Maja Adamska, Alina Craigie, Sandie M Degnan, Bryony Fahey, Marie Gauthier, John N A Hooper, Claire Larroux, Sally P Leys, Erica Lovas, Gemma S Richards

{"title":"The Demosponge Amphimedon queenslandica: Reconstructing the Ancestral Metazoan Genome and Deciphering the Origin of Animal Multicellularity.","authors":"Bernard M Degnan, Maja Adamska, Alina Craigie, Sandie M Degnan, Bryony Fahey, Marie Gauthier, John N A Hooper, Claire Larroux, Sally P Leys, Erica Lovas, Gemma S Richards","doi":"10.1101/pdb.emo108","DOIUrl":"https://doi.org/10.1101/pdb.emo108","url":null,"abstract":"INTRODUCTIONSponges are one of the earliest branching metazoans. In addition to undergoing complex development and differentiation, they can regenerate via stem cells and can discern self from nonself (\"allorecognition\"), making them a useful comparative model for a range of metazoan-specific processes. Molecular analyses of these processes have the potential to reveal ancient homologies shared among all living animals and critical genomic innovations that underpin metazoan multicellularity. Amphimedon queenslandica (Porifera, Demospongiae, Haplosclerida, Niphatidae) is the first poriferan representative to have its genome sequenced, assembled, and annotated. Amphimedon exemplifies many sessile and sedentary marine invertebrates (e.g., corals, ascidians, bryozoans): They disperse during a planktonic larval phase, settle in the vicinity of conspecifics, ward off potential competitors (including incompatible genotypes), and ensure that brooded eggs are fertilized by conspecific sperm. Using genomic and expressed sequence tag (EST) resources from Amphimedon, functional genomic approaches can be applied to a wide range of ecological and population genetic processes, including fertilization, dispersal, and colonization dynamics, host-symbiont interactions, and secondary metabolite production. Unlike most other sponges, Amphimedon produce hundreds of asynchronously developing embryos and larvae year-round in distinct, easily accessible brood chambers. Embryogenesis gives rise to larvae with at least a dozen cell types that are segregated into three layers and patterned along the body axis. In this article, we describe some of the methods currently available for studying A. queenslandica, focusing on the analysis of embryos, larvae, and post-larvae.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2008-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.emo108","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29701408","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: 33

Dictyostelium discoideum: The Social Ameba. 盘状盘骨:社会性阿米巴。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.emo109

Pascale Gaudet, Petra Fey, Rex Chisholm

{"title":"Dictyostelium discoideum: The Social Ameba.","authors":"Pascale Gaudet, Petra Fey, Rex Chisholm","doi":"10.1101/pdb.emo109","DOIUrl":"https://doi.org/10.1101/pdb.emo109","url":null,"abstract":"INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a \"social ameba\" because it can form a multicellular structure when nutrient conditions are limiting. D. discoideum and related organisms, known as the Dictyostelia, have been studied for almost 150 years. The cellular and molecular aspects of their multicellular lifestyle have been studied in detail, and general principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from this work and have been found to be conserved across all eukaryotes. The bacteriovore nature of the unicellular stage provides an excellent model in which to study phagocytosis and the mechanisms of bacterial virulence. D. discoideum has also been used successfully to explore the molecular basis of various human diseases, as well as the mechanisms of drug action and the pathways that lead to resistance to certain therapeutic agents. The availability of a complete genome sequence has further widened the scope of studies using D. discoideum. A large potential for secondary metabolism has become apparent, which opens the door to discovering new compounds with potential medical applications. Numerous putative orthologs of genes responsible for diseases in humans, but whose molecular functions are still uncharacterized, are present in the D. discoideum genome. Finally, the availability of community resources, including the genome database dictyBase and the Dicty Stock Center, makes D. discoideum an easily accessible and powerful model organism to study.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2008-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.emo109","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29701409","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

Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein-Protein Interactions: General Guidelines. 使用核酸可编程蛋白阵列(NAPPA)鉴定蛋白质-蛋白质相互作用:一般指南。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.ip62

Andrew J Link, Joshua Labaer

{"title":"Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein-Protein Interactions: General Guidelines.","authors":"Andrew J Link, Joshua Labaer","doi":"10.1101/pdb.ip62","DOIUrl":"https://doi.org/10.1101/pdb.ip62","url":null,"abstract":"INTRODUCTIONThe Nucleic Acid Programmable Protein Array (NAPPA) approach for producing protein microarrays uses cell-free extracts to transcribe and translate cDNAs encoding target proteins directly onto glass slides. Following array preparation, the identification of protein interactions using NAPPA can be accomplished by either of two general schemas. The first is to probe an expressed NAPPA slide with a purified protein of interest (the query protein) and look for interactors. Signals of these interactions can be detected either by directly labeling the query protein or by using a labeled antibody either to the protein itself or to a tag on the protein. This approach works well when there is access to the purified protein, and it has the advantage that users can test query protein binding with and without post-translational modifications or under a variety of binding conditions. The second schema entails coexpressing the query protein on the NAPPA slide at the same time that all the target proteins are expressed. This article describes the advantages of using a coexpressed query protein and provides advice on choosing a suitable epitope tag.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2008-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.ip62","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29701367","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 amphimedon developmental material. 两栖动物发育物质的分离。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.prot5095

Sally P Leys, Claire Larroux, Marie Gauthier, Maja Adamska, Bryony Fahey, Gemma S Richards, Sandie M Degnan, Bernard M Degnan

引用次数: 31

Analysis of cell movement in amphimedon embryos by injection of fluorescent tracers. 注射荧光示踪剂对两栖动物胚胎细胞运动的分析。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.prot5097

Maja Adamska, Bernard M Degnan

引用次数: 5

Making permanent stocks of dictyostelium. 永久储存盘基骨菌。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.prot5101

Pascale Gaudet, Petra Fey, Rex Chisholm

{"title":"Making permanent stocks of dictyostelium.","authors":"Pascale Gaudet, Petra Fey, Rex Chisholm","doi":"10.1101/pdb.prot5101","DOIUrl":"https://doi.org/10.1101/pdb.prot5101","url":null,"abstract":"INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrients are depleted from the immediate environment of the cells. Dictyostelium can be grown axenically or in the presence of bacteria, either on agar plates or in suspension. Because Dictyostelium growth rates are relatively slow compared to those of bacteria or yeast, laboratories commonly maintain stocks of growing cultures in order to start experiments rapidly. However, it is important to remember that the genome of Dictyostelium, like that of any living organism, is subject to genetic modification. It is well documented that cell lines that are kept in culture for an extended period of time exhibit undesirable changes that yield unreliable experimental results (Hughes et al. 2007). Dictyostelium strains from different laboratories are known to contain various large genome duplications, presumably due to clone selection. Thus, good handling of the cells is essential. To obtain consistent results, new cultures must be started every 2-4 wk, and cultures should never be allowed to grow beyond 4 × 10(6) cells/mL. If overgrowth occurs, a new culture should be started. This protocol describes two methods for preparing long-term stocks of Dictyostelium, either as frozen cells or as spores.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2008-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.prot5101","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29703010","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

Salt gradient dialysis reconstitution of nucleosomes. 核小体的盐梯度透析重构。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.prot5113

Craig L Peterson

引用次数: 9

Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein-Protein Interactions. Protocol 2: Detection of Query Proteins on NAPPA Slides. 利用核酸可编程蛋白阵列(NAPPA)鉴定蛋白-蛋白相互作用。方案2:在NAPPA载玻片上检测查询蛋白。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.prot5109

Andrew J Link, Joshua Labaer

引用次数: 1

Chicken erythrocyte histone octamer preparation. 鸡红细胞组蛋白八聚体制备。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.prot5112

Craig L Peterson, Jeffrey C Hansen

引用次数: 12