The Mitotic Spindle: Methods and Protocols by Paul Chang and Ryoma Ohi


14589beecfe56cb-261x361.jpg Author Paul Chang and Ryoma Ohi
Isbn 9781493935406
File size 13MB
Year 2016
Pages 424
Language English
File format PDF
Category biology


 

METHODS IN MOLECULAR BIOLOGY Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 The Mitotic Spindle Methods and Protocols Edited by Paul Chang Massachusetts Institute of Technology, Cambridge, MA, USA Ryoma Ohi Department of Cell & Developmental Biology, Vanderbilt University, Nashville, TN, USA Editors Paul Chang Massachusetts Institute of Technology Cambridge, MA, USA Ryoma Ohi Department of Cell & Developmental Biology Vanderbilt University Nashville, TN, USA ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-3540-6 ISBN 978-1-4939-3542-0 (eBook) DOI 10.1007/978-1-4939-3542-0 Library of Congress Control Number: 2016938221 © Springer Science+Business Media New York 2016 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC New York Preface The mitotic spindle is a large macromolecular structure that is both beautiful and functionally important. It has long been a focus of intense investigation due to its role as a guardian of ploidy, responsible for the equal segregation of genetic material into daughter cells during cell division. Not surprisingly, the spindle has also been a target of therapeutic inhibition for diseases that involve misregulation of cell division, such as cancer. Indeed, inhibitors of spindle function, such as tubulin poisons, are among the earliest-developed forms of cancer therapy and remain first-line therapies for many forms of cancer. More recently, the spindle has been shown to play equally important roles in other aspects of cell function and human disease. These advances have relied on the development of advanced technology, and as the tools used to study the spindle have evolved so has our understanding of it. Innovations in many areas have impacted mitosis research. Modern mitosis studies began in the 1950s, alongside key developments in light microscopy and the introduction of the electron microscope. Improvements in light microscopy facilitated real-time analysis of cell division and demonstrated the dynamic nature of mitosis. Electron microscopy revealed ultrastructural details of the dividing cell that were critical to the identification of key functional components of the spindle, including the kinetochore. Biochemical discoveries resulted in the identification of tubulin as the building block of the mitotic spindle. Further studies then identified key proteins important for the regulation of microtubule function and have derived key principles of microtubule polymerization dynamics, both of which are fundamental to mitosis. “In vitro” reconstitution of spindle assembly using Xenopus laevis egg extracts provided a simple yet effective assay to identify key components of the spindle required for function. This led to the realization that physics, as well as biology, is central to spindle assembly and the coordination of spindle function. Lastly, knowledge of core cell cycle control mechanisms has allowed us to begin to connect mechanical aspects of mitosis with the physiological state of the cell. Today, methods to analyze mitotic spindle assembly and function are impressively sophisticated and draw from diverse disciplines that include physics, chemistry, and computational modeling. Protein labeling techniques allow us to visualize key spindle components with high spatial and temporal resolution. Chemically induced dimerization gives us control over when and where proteins interact within the cell. Improvements in the isolation and biochemical reconstitution of complex protein assemblies have enabled a thorough documentation of their components and are permitting powerful activity-based studies. In this volume of Methods in Molecular Biology, we have collected a series of protocols from leading investigators in the field whose expertise centers on mitotic spindle function. These methods cover a broad range of techniques, from basic microscopy to the study of spindle functions relevant to cancer. We include methods that can be applied to diverse model systems, which range from the cell-free Xenopus egg extract system to the moss Physcomitrella patens, in an effort to demonstrate the key contributions made by researchers v vi Preface using multiple model organisms. Finally, our chapters integrate cutting-edge technologies, which have only become available due to cross-disciplinary efforts, e.g., analog-sensitive inhibition of kinases. It is our hope that these chapters will be informative for researchers new to mitosis, as well as for those that are already expert in the field. Cambridge, MA, USA Nashville, TN, USA Paul Chang Ryoma Ohi Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PART I v xi METHODS FOCUSED ON CYTOLOGY 1 Using Fluorescence Microscopy to Study Mitosis . . . . . . . . . . . . . . . . . . . . . . Sai K. Balchand, Barbara J. Mann, and Patricia Wadsworth 2 Using Photoactivatable GFP to Study Microtubule Dynamics and Chromosome Segregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bin He and Daniela Cimini 3 15 PART II METHODS FOCUSED ON MICROTUBULES AND THE MITOTIC SPINDLE 3 Purification and Fluorescent Labeling of Tubulin from Xenopus laevis Egg Extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aaron C. Groen and Timothy J. Mitchison 4 Measuring the Effects of Microtubule-Associated Proteins on Microtubule Dynamics In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Marija Zanic 5 Imaging and Quantifying the Dynamics of γ-Tubulin at Microtubule Minus Ends in Mitotic Spindles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nicolas Lecland and Jens Lüders 6 Visualizing and Analyzing Branching Microtubule Nucleation Using Meiotic Xenopus Egg Extracts and TIRF Microscopy . . . . . . . . . . . . . . Matthew King and Sabine Petry 7 Encapsulation of Xenopus Egg and Embryo Extract Spindle Assembly Reactions in Synthetic Cell-Like Compartments with Tunable Size . . . . . . . . . Matthew C. Good PART III 35 47 63 77 87 METHODS FOCUSED ON KINETOCHORES KINETOCHORE-MICROTUBULE INTERFACE AND THE 8 In Vitro Kinetochore Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Matthew D.D. Miell and Aaron F. Straight 9 Biochemical and Structural Analysis of Kinetochore Histone-Fold Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tatsuya Nishino and Tatsuo Fukagawa 10 Measuring Kinetochore–Microtubule Attachment Stability in Cultured Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Keith F. DeLuca, Jacob A. Herman, and Jennifer G. DeLuca vii 111 135 147 viii Contents 11 Studying Kinetochores In Vivo Using FLIM-FRET. . . . . . . . . . . . . . . . . . . . . Tae Yeon Yoo and Daniel J. Needleman PART IV METHODS FOCUSED ON THE SPINDLE POLE 12 Purification of Fluorescently Labeled Saccharomyces cerevisiae Spindle Pole Bodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kimberly K. Fong, Beth Graczyk, and Trisha N. Davis 13 A Cell-Free System for Real-Time Analyses of Centriole Disengagement and Centriole-to-Centrosome Conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rajesh Kumar Soni and Meng-Fu Bryan Tsou 14 Assays to Study Mitotic Centrosome and Spindle Pole Assembly and Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vladimir Joukov, Johannes C. Walter, and Arcangela De Nicolo PART V 169 189 197 207 METHODS FOCUSED ON THE CELLULAR FUNCTIONS MICROTUBULE MOTOR PROTEINS OF 15 Analyzing Spindle Positioning Dynamics in Cultured Cells . . . . . . . . . . . . . . . Tomomi Kiyomitsu 16 Quantification of Mitotic Chromosome Alignment . . . . . . . . . . . . . . . . . . . . . Cindy Fonseca and Jason Stumpff 17 Imaging Mitosis in the Moss Physcomitrella patens. . . . . . . . . . . . . . . . . . . . . . Moé Yamada, Tomohiro Miki, and Gohta Goshima 18 Small Molecule Approach to Study the Function of Mitotic Kinesins . . . . . . . . Naowras Al-Obaidi, Johanna Kastl, and Thomas U. Mayer PART VI 253 263 283 NOVEL APPROACHES TO STUDY SPINDLE FUNCTION AND REGULATION 19 Identification and Characterization of Mitotic Spindle-Localized Transcripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amy B. Emerman, Ashwini Jambhekar, and Michael D. Blower 20 Probing Mitosis by Manipulating the Interactions of Mitotic Regulator Proteins Using Rapamycin-Inducible Dimerization . . . . . . . . . . . . . . . . . . . . . Edward R. Ballister and Michael A. Lampson 21 Studying Kinetochore Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adrian T. Saurin and Geert J.P.L. Kops 22 Engineering and Functional Analysis of Mitotic Kinases Through Chemical Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mathew J.K. Jones and Prasad V. Jallepalli PART VII 239 303 325 333 349 THE MITOTIC SPINDLE AND CANCER 23 Using Cell Culture Models of Centrosome Amplification to Study Centrosome Clustering in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . Mijung Kwon 367 Contents ix 24 Generation and Purification of Tetraploid Cells . . . . . . . . . . . . . . . . . . . . . . . . Elizabeth M. Shenk and Neil J. Ganem 25 Anti-Microtubule Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stefan Florian and Timothy J. Mitchison 393 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423 403 Contributors NAOWRAS AL-OBAIDI • Department of Biology, Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Konstanz, Germany SAI K. BALCHAND • Department of Biology and Program in Molecular and Cellular Biology, University of Massachusetts Amherst, Amherst, MA, USA EDWARD R. BALLISTER • Department of Biology, University of Pennsylvania, Philadelphia, PA, USA MICHAEL D. BLOWER • Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA; Department of Genetics, Harvard Medical School, Boston, MA, USA DANIELA CIMINI • Department of Biological Sciences, Biocomplexity Institute, Virginia Tech, Blacksburg, VA, USA TRISHA N. DAVIS • Department of Biochemistry, University of Washington, Seattle, WA, USA JENNIFER G. DELUCA • Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA KEITH F. DELUCA • Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA AMY B. EMERMAN • Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA; Department of Genetics, Harvard Medical School, Boston, MA, USA STEFAN FLORIAN • Department of Systems Biology, Harvard Medical School, Boston, MA, USA KIMBERLY K. FONG • Department of Biochemistry, University of Washington, Seattle, WA, USA CINDY FONSECA • Department of Molecular Physiology and Biophysics, University of Vermont College of Medicine, Burlington, VT, USA TATSUO FUKAGAWA • Department of Molecular Genetics, National Institute of Genetics and Graduate University for Advanced Studies (SOKENDAI), Shizuoka, Japan; Graduate School of Frontier Biosciences, Osaka University, Suita, Japan NEIL J. GANEM • Division of Hematology and Oncology, Department of Pharmacology, Boston University School of Medicine, Boston, MA, USA; Department of Experimental Therapeutics and Medicine, Boston University School of Medicine, Boston, MA, USA MATTHEW C. GOOD • Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA; Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA; Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, PA, USA GOHTA GOSHIMA • Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan BETH GRACZYK • Department of Biochemistry, University of Washington, Seattle, WA, USA AARON C. GROEN • Department of Systems Biology, Harvard Medical School, Boston, MA, USA BIN HE • Department of Biological Sciences, Biocomplexity Institute, Virginia Tech, Blacksburg, VA, USA JACOB A. HERMAN • Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA xi xii Contributors PRASAD V. JALLEPALLI • Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA ASHWINI JAMBHEKAR • Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA; Department of Genetics, Harvard Medical School, Boston, MA, USA MATHEW J.K. JONES • Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA VLADIMIR JOUKOV • Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA JOHANNA KASTL • Department of Biology, Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Konstanz, Germany MATTHEW KING • Department of Molecular Biology, Princeton University, Princeton, NJ, USA TOMOMI KIYOMITSU • Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan; Precursory Research for Embryonic Science and Technology (PRESTO) Program, Japan Science and Technology Agency, Saitama, Japan GEERT J.P.L. KOPS • Hubrecht Institute – KNAW (Royal Netherlands Academy of Arts and Sciences), Utrecht, The Netherlands; Cancer Genomics Netherlands, University Medical Center Utrecht, Utrecht, The Netherlands MIJUNG KWON • Department of Pediatric Oncology, Howard Hughes Medical Institute, Dana-Farber Cancer Institute, Boston, MA, USA; Department of Cell Biology, Harvard Medical School, Boston, MA, USA MICHAEL A. LAMPSON • Department of Biology, University of Pennsylvania, Philadelphia, PA, USA NICOLAS LECLAND • Centre Biologie du Développement, UMR 5547 CNRS-Université Paul Sabatier, Toulouse, France JENS LÜDERS • Cell and Developmental Biology Programme, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain BARBARA J. MANN • Department of Biology and Program in Molecular and Cellular Biology, University of Massachusetts Amherst, Amherst, MA, USA THOMAS U. MAYER • Department of Biology, Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Konstanz, Germany MATTHEW D.D. MIELL • Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA TOMOHIRO MIKI • Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan TIMOTHY J. MITCHISON • Department of Systems Biology, Harvard Medical School, Boston, MA, USA DANIEL J. NEEDLEMAN • School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA; Departments of Applied Physics, and Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA ARCANGELA DE NICOLO • Department of Medicine, Harvard Medical School, Boston, MA, USA; Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA TATSUYA NISHINO • Department of Molecular Genetics, National Institute of Genetics and Graduate University for Advanced Studies (SOKENDAI), Shizuoka, Japan; Department of Biological Science and Technology, Graduate School of Industrial Science and Technology, Tokyo University of Science, Tokyo, Japan SABINE PETRY • Department of Molecular Biology, Princeton University, Princeton, NJ, USA Contributors xiii ADRIAN T. SAURIN • Division of Cancer Research, Medical Research Institute, Jacqui Wood Cancer Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK ELIZABETH M. SHENK • Division of Hematology and Oncology, Department of Pharmacology, Boston University School of Medicine, Boston, MA, USA; Department of Experimental Therapeutics and Medicine, Boston University School of Medicine, Boston, MA, USA RAJESH KUMAR SONI • Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA AARON F. STRAIGHT • Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA JASON STUMPFF • Department of Molecular Physiology and Biophysics, University of Vermont College of Medicine, Burlington, VT, USA MENG-FU BRYAN TSOU • Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA PATRICIA WADSWORTH • Department of Biology, Program in Molecular and Cellular Biology, University of Massachusetts Amherst, Amherst, MA, USA JOHANNES C. WALTER • Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Howard Hughes Medical Institute, Boston, MA, USA MOÉ YAMADA • Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan TAE YEON YOO • School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA MARIJA ZANIC • Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA; Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA Part I Methods Focused on Cytology Chapter 1 Using Fluorescence Microscopy to Study Mitosis Sai K. Balchand, Barbara J. Mann, and Patricia Wadsworth Abstract Fluorescence microscopy is one of the most important approaches in the cell biologist’s toolbox for studying the mitotic spindle. In fact, many of the key insights into our understanding of mitosis have been enabled by the visualization of mitotic processes using fluorescence microscopy. Here, we summarize some of the important considerations for imaging mitosis using fluorescence microscopy. Because light can damage live cells, we emphasize the importance of minimizing cellular damage while obtaining informative images. Key words Spinning disc confocal microscopy, Confocal laser scanning microscopy, Mitosis 1 Introduction The impact of fluorescence microscopy on cell biology in general, and on the field of mitosis in particular, is profound. This major impact has occurred due to the confluence of the development of fluorescent reporter molecules that can be delivered to and expressed in live cells and advances in equipment used for imaging. Over the past few decades, major improvements in detectors, light sources, filters, objective lenses, and the increased availability of turnkey systems for confocal microscopy have provided powerful tools to the researcher seeking to image the events of mitosis. Researchers have taken advantage of sophisticated new equipment to image cells that have been engineered to express mitotic proteins tagged with genetically encoded fluorescent proteins, FRET sensors and other fluorescent markers. Methods for introducing engineered proteins into cells have been greatly improved, enabling the researcher to control level of expression [1] and to perform near simultaneous imaging of multiple fluorescent proteins in the spindle. Moreover, recent introduction of gene editing protocols for mammalian cells promises to revolutionize imaging of tagged proteins by permitting modification of the endogenous genetic Paul Chang and Ryoma Ohi (eds.), The Mitotic Spindle: Methods and Protocols, Methods in Molecular Biology, vol. 1413, DOI 10.1007/978-1-4939-3542-0_1, © Springer Science+Business Media New York 2016 3 4 Sai K. Balchand et al. locus with fluorescent tags [2]. These new developments will undoubtedly provide unanticipated insights into mitosis. Obtaining meaningful, quantitative data with the fluorescence light microscope requires a thorough understanding of the many variables that must be controlled during imaging [3]. Perhaps the most important consideration is that the cells must be kept healthy and dividing throughout the acquisition process [4]. Unfortunately, light is not inert and will cause damage to the live cells if not used judiciously. Fortunately, many key parameters of the experimental design for successful live cell imaging using fluorescence microscopy have been worked out [5]; similarly, protocols for generating cell lines expressing tagged proteins are also available [6, 7]. Here, we outline methods for fluorescence microscopy of mitosis in mammalian cultured cells, including maintaining cell viability. With minor modifications, similar methods can also be used for other cultured cells from other species (e.g., Drosophila S2 cells) and other model systems (e.g., C. elegans embryos). 2 Materials 1. Non-CO2 medium: culture medium without phenol red indicator dye and without bicarbonate buffer and containing 20 mM HEPES, pH adjusted to 7.2 with potassium hydroxide, and supplemented with 10 % fetal bovine serum (FBS). 2. Oxygen scavenging system, e.g., Oxyrase. 3. Sterile glass coverslips, thickness #1.5. 4. Chambers for holding coverslips or glass bottom culture dishes. 5. Thermistor probe. 6. Vacuum grease or mineral oil. 7. Lens cleaner. 3 Methods 3.1 Maintenance of Mitotic Cell Health It is of utmost importance that cell health is maintained during imaging. In the case of imaging mitotic cells, this can be easily evaluated by comparing the progress through mitosis of the imaged cells with appropriate control cells that were not exposed to fluorescent light. Successful completion of mitosis, with kinetics similar to the control cells, is a good indication that the imaging protocol has not caused cellular damage. Whenever possible, mitotic progression should be evaluated for the imaging conditions used for your experiments. Fluorescence Microscopy of Mitotic Cells 3.1.1 Protocol 1: Maintaining Mitotic Progression During Imaging 5 1. Plate the cells in appropriate dishes or on coverslips for use in closed chambers (see Note 1). Coverslips should be flamesterilized or autoclaved prior to use; #1.5 glass is the proper thickness for high numerical aperture objective lenses (see Note 2). Cells are typically plated 1–2 days prior to experimentation. 2. Choose an appropriate stage/microscope heater system and pre-warm to the desired temperature (see Note 3). 3. Remove the cell culture medium and replace with pre-warmed Non-CO2 medium that lacks bicarbonate buffer and contains 20 mM HEPES, pH 7.2 with potassium hydroxide, and supplemented with 10 % FBS. Non-CO2 culture medium lacks phenol red, which is autofluorescent. 4. Add an oxygen scavenging system to retard photobleaching. Because Oxyrase contains bacterial membrane fragments, it is added to the medium, and then filtered, using a 0.22 μm syringe filter, prior to adding to the chamber or dish containing the cells. 5. Bring the cells to the microscope for imaging. 3.2 Fluorescence Microscopy of Mitotic Cells 3.2.1 Choosing an Imaging System 3.2.2 Protocol 2: Imaging Mitosis Using Spinning Disc Confocal Microscopy For a number of years, imaging mitotic cells by spinning disc confocal microscopy (SDCM) has been the gold standard in the field, primarily because the rapid acquisition possible with this type of confocal system enables the dynamics of the mitotic spindle to be captured [8]. However, laser scanning confocal microscopes (LSCM) can also capture dynamic events, especially instruments equipped with resonant scanning mirrors. LSCMs have additional advantages, including the ability to change pinhole size, to increase magnification using electronic zoom, spectral detectors to enable imaging with a wide range of fluorophores and built-in capacity for photobleaching and photoactivation experiments [3]. For a discussion of some of the major differences between these types of confocal systems, and the option of imaging using wide-field fluorescence followed by deconvolution, see Note 4. 1. Turn on microscope system and follow the manufacturer’s recommendation for warming up the lasers. This protocol assumes familiarity with the basic software commands for the system you are using. 2. Bring a dish of cells to the microscope in pre-warmed nonCO2 medium and secure the dish to the microscope stage. Because cells are not under ideal conditions, i.e., they are in a non-CO2 buffered environment, switch to a new dish of cells after about 1 h of imaging. Long-term imaging is best accomplished with an environmental chamber that controls humidity, gas, and temperature.

Author Paul Chang and Ryoma Ohi Isbn 9781493935406 File size 13MB Year 2016 Pages 424 Language English File format PDF Category Biology Book Description: FacebookTwitterGoogle+TumblrDiggMySpaceShare This volume includes a series of protocols focused on mitotic spindle assembly and function. The methods covered in this book feature a broad range of techniques from basic microscopy to the study of spindle physiologies relevant to cancer. These methods can be applied to diverse model systems that range from the cell-free Xenopus egg extract system to the moss Physcomitrella patens, in an effort to demonstrate the key contributions made by researchers using multiple model organisms. Chapters in The Mitotic Spindle: Methods and Protocolsintegrate cutting-edge technologies that have only become available due to the cross-disciplinary efforts, such as ATP analogue sensitive inhibition of mitotic kinases. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Thorough and informative, The Mitotic Spindle: Methods and Protocols, is a valuable resource for researchers who are new to mitosis or are already experts in the field.       Download (13MB) Tumor Angiogenesis Assays: Methods and Protocols Protein Chromatography: Methods and Protocols, 2nd ed. Gene Regulation: Methods And Protocols Zebrafish: Methods and Protocols, 2nd Edition Synthetic Protein Switches: Methods and Protocols Load more posts

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