Current Protocols in Cell Biology最新文献_第10页

Cellular Detection of G-Quadruplexes by Optical Imaging Methods. 光学成像方法检测g -四联体细胞。

Current Protocols in Cell Biology Pub Date : 2017-09-01 DOI: 10.1002/cpcb.29

Souheila Amor, Sunny Y Yang, Judy M Y Wong, David Monchaud

{"title":"Cellular Detection of G-Quadruplexes by Optical Imaging Methods.","authors":"Souheila Amor, Sunny Y Yang, Judy M Y Wong, David Monchaud","doi":"10.1002/cpcb.29","DOIUrl":"https://doi.org/10.1002/cpcb.29","url":null,"abstract":"G-quadruplexes (G4s) are higher-order nucleic acid structures that fold from guanine (G)-rich DNA and RNA strands. This field of research gains traction as a major chemical biology area since it aims at uncovering many key cellular mechanisms in which quadruplexes are involved. The wealth of knowledge acquired over the past three decades strongly supports pivotal roles of G4 in the regulation of gene expression at both transcriptional (DNA quadruplexes) and translational levels (RNA quadruplexes). Recent biochemical discoveries uncovered myriad of additional G4 actions: from chromosomal stability to the firing of replication origins, from telomere homeostasis to functional dysregulations underlying genetic diseases (including cancers and neurodegeneration). Here, we listed a repertoire of protocols that we have developed over the past years to visualize quadruplexes in cells. These achievements were made possible thanks to the discovery of a novel family of versatile quadruplex-selective fluorophores, the twice-as-smart quadruplex ligands named TASQ (for template-assembled synthetic G-quartet). The versatility of this probe allows for multiple imaging techniques in both fixed and live cells, including the use of the multiphoton microscopy, confocal microscopy, and real-time fluorescent image collection. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"76 ","pages":"4.33.1-4.33.19"},"PeriodicalIF":0.0,"publicationDate":"2017-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.29","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35464949","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

Determination of Membrane Protein Distribution on the Nuclear Envelope by Single-Point Single-Molecule FRAP. 单点单分子FRAP法测定核膜上膜蛋白分布。

Current Protocols in Cell Biology Pub Date : 2017-09-01 DOI: 10.1002/cpcb.27

Krishna C Mudumbi, Weidong Yang

引用次数: 1

Lectin-Array Blotting. Lectin-Array印迹。

Current Protocols in Cell Biology Pub Date : 2017-09-01 DOI: 10.1002/cpcb.20

Raquel Pazos, Juan Echevarria, Alvaro Hernandez, Niels-Christian Reichardt

{"title":"Lectin-Array Blotting.","authors":"Raquel Pazos, Juan Echevarria, Alvaro Hernandez, Niels-Christian Reichardt","doi":"10.1002/cpcb.20","DOIUrl":"https://doi.org/10.1002/cpcb.20","url":null,"abstract":"Aberrant protein glycosylation is a hallmark of cancer, infectious diseases, and autoimmune or neurodegenerative disorders. Unlocking the potential of glycans as disease markers will require rapid and unbiased glycoproteomics methods for glycan biomarker discovery. The present method is a facile and rapid protocol for qualitative analysis of protein glycosylation in complex biological mixtures. While traditional lectin arrays only provide an average signal for the glycans in the mixture, which is usually dominated by the most abundant proteins, our method provides individual lectin binding profiles for all proteins separated in the gel electrophoresis step. Proteins do not have to be excised from the gel for subsequent analysis via the lectin array but are transferred by contact diffusion from the gel to a glass slide presenting multiple copies of printed lectin arrays. Fluorescently marked glycoproteins are trapped by the printed lectins via specific carbohydrate-lectin interactions and after a washing step their binding profile with up to 20 lectin probes is analyzed with a fluorescent scanner. The method produces the equivalent of 20 lectin blots in a single experiment, giving detailed insight into the binding epitopes present in the fractionated proteins. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"76 ","pages":"6.12.1-6.12.12"},"PeriodicalIF":0.0,"publicationDate":"2017-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.20","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35464948","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

Centrifugation-Free Magnetic Isolation of Functional Mitochondria Using Paramagnetic Iron Oxide Nanoparticles. 顺磁性氧化铁纳米颗粒无离心磁分离功能线粒体。

Current Protocols in Cell Biology Pub Date : 2017-09-01 DOI: 10.1002/cpcb.26

Bhabatosh Banik, Shanta Dhar

引用次数: 10

Automated Tracking of Cell Migration with Rapid Data Analysis. 快速数据分析的细胞迁移自动跟踪。

Current Protocols in Cell Biology Pub Date : 2017-09-01 DOI: 10.1002/cpcb.28

Brian J DuChez

{"title":"Automated Tracking of Cell Migration with Rapid Data Analysis.","authors":"Brian J DuChez","doi":"10.1002/cpcb.28","DOIUrl":"https://doi.org/10.1002/cpcb.28","url":null,"abstract":"Cell migration is essential for many biological processes including development, wound healing, and metastasis. However, studying cell migration often requires the time-consuming and labor-intensive task of manually tracking cells. To accelerate the task of obtaining coordinate positions of migrating cells, we have developed a graphical user interface (GUI) capable of automating the tracking of fluorescently labeled nuclei. This GUI provides an intuitive user interface that makes automated tracking accessible to researchers with no image-processing experience or familiarity with particle-tracking approaches. Using this GUI, users can interactively determine a minimum of four parameters to identify fluorescently labeled cells and automate acquisition of cell trajectories. Additional features allow for batch processing of numerous time-lapse images, curation of unwanted tracks, and subsequent statistical analysis of tracked cells. Statistical outputs allow users to evaluate migratory phenotypes, including cell speed, distance, displacement, and persistence, as well as measures of directional movement, such as forward migration index (FMI) and angular displacement. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"76 ","pages":"12.12.1-12.12.16"},"PeriodicalIF":0.0,"publicationDate":"2017-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.28","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35317200","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 Vitro Reconstitution of the Endoplasmic Reticulum. 内质网的体外重建。

Current Protocols in Cell Biology Pub Date : 2017-09-01 DOI: 10.1002/cpcb.30

Csilla-Maria Ferencz, Gernot Guigas, Andreas Veres, Brigitte Neumann, Olaf Stemmann, Matthias Weiss

{"title":"In Vitro Reconstitution of the Endoplasmic Reticulum.","authors":"Csilla-Maria Ferencz, Gernot Guigas, Andreas Veres, Brigitte Neumann, Olaf Stemmann, Matthias Weiss","doi":"10.1002/cpcb.30","DOIUrl":"https://doi.org/10.1002/cpcb.30","url":null,"abstract":"Reconstitution of cellular organelles in vitro offers the possibility to perform quantitative and qualitative experiments in a controlled environment that cannot be done with the same accuracy in living cells. Following a previous report, the subsequent list of protocols describes how to reconstitute and quantify a tubular ER network in vitro based on purified microsomes from culture cells and cytosol from Xenopus laevis egg extracts. Biological material preparation and reconstitution assays require mostly basic laboratory instrumentation and chemicals, and can be executed without any specific training, making them appealing to a wide range of laboratories. Moreover, to promote conditions that are markedly more reflective of in vivo environments, this method describes for the first time in the literature, the purification of microsomes from HeLa cells in some detail. Basic Protocol 1 in this article describes the reconstitution process on different substrates including the associated fluorescence imaging process. Purification of ER microsomes and cytosol, both of which are needed for this approach, are described in detail in Support Protocols 1 and 2, respectively. Coating of surfaces with polyacrylamide gels is described in Support Protocol 3. Basic Protocol 2 outlines how to segment and skeletonize fluorescence images of ER networks, and how to quantify segment lengths between the network's branching points. The described quantitative evaluation provides a meaningful approach to analyze the topology and geometry of organelle structures. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"76 ","pages":"11.22.1-11.22.16"},"PeriodicalIF":0.0,"publicationDate":"2017-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.30","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35464945","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

Labeling and Magnetic Resonance Imaging of Exosomes Isolated from Adipose Stem Cells 脂肪干细胞外泌体的标记和磁共振成像

Current Protocols in Cell Biology Pub Date : 2017-06-19 DOI: 10.1002/cpcb.23

Alice Busato, Roberta Bonafede, Pietro Bontempi, Ilaria Scambi, Lorenzo Schiaffino, Donatella Benati, Manuela Malatesta, Andrea Sbarbati, Pasquina Marzola, Raffaella Mariotti

{"title":"Labeling and Magnetic Resonance Imaging of Exosomes Isolated from Adipose Stem Cells","authors":"Alice Busato, Roberta Bonafede, Pietro Bontempi, Ilaria Scambi, Lorenzo Schiaffino, Donatella Benati, Manuela Malatesta, Andrea Sbarbati, Pasquina Marzola, Raffaella Mariotti","doi":"10.1002/cpcb.23","DOIUrl":"10.1002/cpcb.23","url":null,"abstract":"Adipose stem cells (ASC) represent a promising therapeutic approach for neurodegenerative diseases. Most biological effects of ASC are probably mediated by extracellular vesicles, such as exosomes, which influence the surrounding cells. Current development of exosome therapies requires efficient and noninvasive methods to localize, monitor, and track the exosomes. Among imaging methods used for this purpose, magnetic resonance imaging (MRI) has advantages: high spatial resolution, rapid in vivo acquisition, and radiation-free operation. To be detectable with MRI, exosomes must be labeled with MR contrast agents, such as ultra-small superparamagnetic iron oxide nanoparticles (USPIO). Here, we set up an innovative approach for exosome labeling that preserves their morphology and physiological characteristics. We show that by labeling ASC with USPIO before extraction of nanovesicles, the isolated exosomes retain nanoparticles and can be visualized by MRI. The current work aims at validating this novel USPIO-based exosome labeling method by monitoring the efficiency of the labeling with MRI both in ASC and in exosomes. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"75 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.23","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35098985","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: 39

Labeling DNA Replication Foci to Visualize Chromosome Territories In Vivo 标记DNA复制焦点以可视化体内染色体区域

Current Protocols in Cell Biology Pub Date : 2017-06-19 DOI: 10.1002/cpcb.19

Apolinar Maya-Mendoza, Dean A. Jackson

引用次数: 2

Microcontact Peeling: A Cell Micropatterning Technique for Circumventing Direct Adsorption of Proteins to Hydrophobic PDMS 微接触剥落:一种避免蛋白质在疏水性PDMS上直接吸附的细胞微图技术

Current Protocols in Cell Biology Pub Date : 2017-06-19 DOI: 10.1002/cpcb.22

Sho Yokoyama, Tsubasa S. Matsui, Shinji Deguchi

{"title":"Microcontact Peeling: A Cell Micropatterning Technique for Circumventing Direct Adsorption of Proteins to Hydrophobic PDMS","authors":"Sho Yokoyama, Tsubasa S. Matsui, Shinji Deguchi","doi":"10.1002/cpcb.22","DOIUrl":"10.1002/cpcb.22","url":null,"abstract":"Microcontact printing (μCPr) is one of the most popular techniques used for cell micropatterning. In conventional μCPr, a polydimethylsiloxane (PDMS) stamp with microfeatures is used to adsorb extracellular matrix (ECM) proteins onto the featured surface and transfer them onto particular areas of a cell culture substrate. However, some types of functional proteins other than ECM have been reported to denature upon direct adsorption to hydrophobic PDMS. Here we describe a detailed protocol of an alternative technique––microcontact peeling (μCPe)––that allows for cell micropatterning while circumventing the step of adsorbing proteins to bare PDMS. This technique employs microfeatured materials with a relatively high surface energy such as copper, instead of using a microfeatured PDMS stamp, to peel off a cell-adhesive layer present on the surface of substrates. Consequently, cell-nonadhesive substrates are exposed at the specific surface that undergoes the physical contact with the microfeatured material. Thus, although μCPe and μCPr are apparently similar, the former does not comprise a process of transferring biomolecules through hydrophobic PDMS. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"75 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.22","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35099440","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

Patterning on Topography for Generation of Cell Culture Substrates with Independent Nanoscale Control of Chemical and Topographical Extracellular Matrix Cues 利用独立的纳米尺度控制化学和地形的细胞外基质线索生成细胞培养基质的地形图案

Current Protocols in Cell Biology Pub Date : 2017-06-19 DOI: 10.1002/cpcb.25

Emily N. Sevcik, John M. Szymanski, Quentin Jallerat, Adam W. Feinberg

{"title":"Patterning on Topography for Generation of Cell Culture Substrates with Independent Nanoscale Control of Chemical and Topographical Extracellular Matrix Cues","authors":"Emily N. Sevcik, John M. Szymanski, Quentin Jallerat, Adam W. Feinberg","doi":"10.1002/cpcb.25","DOIUrl":"10.1002/cpcb.25","url":null,"abstract":"The cell microenvironment plays an important role in many biological processes, including development and disease progression. Key to this is the extracellular matrix (ECM), a complex biopolymer network serving as the primary insoluble signaling network for physical, chemical, and mechanical cues. In vitro, the ability to engineer the ECM at the micro- and nanoscales is a critical tool to systematically interrogate the influence of ECM properties on cellular responses. Specifically, both topographical and chemical surface patterning has been shown to direct cell alignment and tissue architecture on biomaterial surfaces, however, it has proven challenging to independently control these surface properties. This protocol describes a method termed Patterning on Topography (PoT) to engineer 2D nanopatterns of ECM proteins onto topographically complex substrates, which enables independent control of physical and chemical surface properties. Applications include interrogation of fundamental cell-surface interactions and engineering interfaces that can direct cell and/or tissue function. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"75 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.25","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35098983","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: 10