University of Edinburgh

57 articles published in JoVE

• Genetics

  
  Sample Preparation to Bioinformatics Analysis of DNA Methylation: Association Strategy for Obesity and Related Trait Studies Natália Yumi Noronha*¹, Guilherme da Silva Rodrigues*¹, Marcela Augusta de Souza Pinhel¹, Jean-Baptiste Cazier^2,3, Lígia Moriguchi Watanabe¹, Albert Nobre Menezes⁴, Carlos Roberto Bueno⁵, Carolina Ferreira Nicoletti¹, Bruno Affonso Parenti de Oliveira¹, Isabelle Mello Schineider⁶, Isabella Harumi Yonehara Noma⁷, Igor Caetano Dias Alcarás⁸, Fernando Barbosa⁹, Carla Barbosa Nonino^{6 1}Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, ²Centre for Computational Biology, University of Birmingham, ³Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, ⁴Cancer Genetics and Evolution Laboratory, Cancer Research UK, Institute of Genetics & Molecular Medicine, The University of Edinburgh, ⁵Ribeirão Preto School of Physical Education and Sport, University of São Paulo, ⁶Health Sciences Department, Ribeirao Preto Medical School, University of Sao Paulo, ⁷Faculty of Pharmaceutical Sciences, University of São Paulo, ⁸Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, ⁹Department of Clinical, Bromatological and Toxicological Analysis, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of São Paulo The present study describes the workflow to manage DNA methylation data obtained by microarray technologies. The protocol demonstrates steps from sample preparation to data analysis. All procedures are described in detail, and the video shows the significant steps.

• Biology

  
  Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System Jose Meseguer-Ripolles¹, Yu Wang¹, Agnes Sorteberg¹, Aishwariya Sharma², Nan-Linda Ding², Baltasar Lucendo-Villarin¹, Philipp Kramer², Charis-Patricia Segeritz², David C. Hay^{1 1}MRC Centre for Regenerative Medicine, University of Edinburgh, ²Research & Development, STEMCELL Technologies Inc The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

• Genetics

  
  Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing Yu Wang¹, Andrew J. H. Smith¹, David C. Hay^{1 1}MRC Centre for Regenerative Medicine, University of Edinburgh Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4α) locus in human embryonic stem cells (hESCs).

• Biochemistry

  
  Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using χCRAC Stuart W. McKellar¹, Ivayla Ivanova¹, Robert W. van Nues², Ross A. Cordiner³, Mehak Chauhan¹, Niki Christopoulou¹, Will Worboys⁴, Andrew Langford⁴, Torben Heick Jensen³, Sander Granneman^{1 1}Centre for Engineering Biology, University of Edinburgh, ²Institute of Cell Biology, University of Edinburgh, ³Department of Molecular Biology and Genetics, Aarhus University, ⁴UVO3 Ltd. Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

• Immunology and Infection

  
  Production and Characterization of Human Macrophages from Pluripotent Stem Cells Martha Lopez-Yrigoyen^1,2, Alisha May¹, Telma Ventura¹, Helen Taylor¹, Antonella Fidanza¹, Luca Cassetta², Jeffrey W. Pollard², Lesley M. Forrester^{1 1}Centre for Regenerative Medicine, Scottish Centre for Regenerative Medicine, University of Edinburgh, ²Centre for Reproductive Health, The Queen's Medical Research Institute, University of Edinburgh This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived macrophages.

• Neuroscience

  
  Three-Dimensional Shape Modeling and Analysis of Brain Structures Jaeil Kim¹, Maria del Carmen Valdés Hernández², Jinah Park^{3 1}School of Computer Science and Engineering, Kyungpook National University, ²Centre for Clinical Brain Sciences, University of Edinburgh, ³School of Computing and KI for Health Science and Technology (KIHST), Korea Advanced Institute of Science and Technology (KAIST) We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

• Biology

  
  Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit Grant A. R. Gale*^1,2,3, Alejandra A. Schiavon Osorio*^1,2, Anton Puzorjov*^1,2, Baojun Wang^2,3, Alistair J. McCormick^{1,2 1}Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh, ²Centre for Synthetic and Systems Biology, University of Edinburgh, ³Institute of Quantitative Biology, Biochemistry and Biotechnology, School of Biological Sciences, University of Edinburgh Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

• Biology

  
  Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization Bianca Vezzani^1,2, Mario Gomez-Salazar¹, Joan Casamitjana¹, Carlo Tremolada³, Bruno Péault^{1,4 1}MRC Center for Regenerative Medicine, University of Edinburgh, ²Dept. of Morphology, Surgery and Experimental Medicine, Section of General Pathology, University of Ferrara, ³Italian Image Institute, ⁴Orthopaedic Hospital Research Center and Broad Stem Cell Research Center, David Geffen School of Medicine, University of California Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.

• Biochemistry

  
  Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU) J. David Barrass¹, Jean D. Beggs^{1 1}Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

• Developmental Biology

  
  Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells Balta Lucendo-Villarin¹, Hassan Rashidi^1,2, Sharmin Alhaque^1,3, Lena Fischer^1,4, Jose Meseguer-Ripolles¹, Yu Wang¹, Cliona O'Farrelly⁴, Michael Themis³, David C. Hay^{1 1}MRC Centre for Regenerative Medicine, University of Edinburgh, ²UCL Great Ormond Street Institute of Child Health, University College London, ³Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, ⁴School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

• Immunology and Infection

  
  Tuning Degradation to Achieve Specific and Efficient Protein Depletion J. David Barrass¹, Gonzalo I. Mendoza-Ochoa^1,2, Isabella E. Maudlin^1,3, Emanuela Sani¹, Jean D. Beggs^{1 1}Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, ²Department of Plant Sciences, University of Cambridge, ³Sir William Dunn School of Pathology, University of Oxford Here, we present a protocol to effectively and specifically deplete a protein of interest in the yeast Saccharomyces cerevisiae using the β-est AID system.

• Developmental Biology

  
  Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging Darren Wisniewski¹, Sally Lowell¹, Guillaume Blin^{1 1}MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

• Biochemistry

  
  Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting Yu-Ting Huang^1,2, Dinja van der Hoorn*^1,2, Leire M. Ledahawsky*^1,2, Anna A. L. Motyl*^1,2, Crispin Y. Jordan¹, Thomas H. Gillingwater^1,2, Ewout J. N. Groen^{1,2 1}Centre for Discovery Brain Sciences, University of Edinburgh, ²Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

• Cancer Research

  
  Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8⁺ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells Takanori Kitamura*^1,2, Dahlia Doughty-Shenton*³, Jeffrey W. Pollard², Neil O. Carragher^{3,4 1}Royal (Dick) School of Veterinary Studies and Roslin Institute, University of Edinburgh, ²MRC Centre for Reproductive Health, University of Edinburgh, ³Edinburgh Phenotypic Assay Centre, University of Edinburgh, ⁴Cancer Research UK Edinburgh Centre, MRC Institute of Genetics, Molecular Medicine, University of Edinburgh We describe here a protocol to investigate cytotoxicity of pre-activated CD8⁺ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

• Developmental Biology

  
  Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells Jose Meseguer-Ripolles¹, Baltasar Lucendo-Villarin¹, Yu Wang¹, David C. Hay^{1 1}MRC Centre for Regenerative Medicine, University of Edinburgh This protocol describes a semi-automated approach to produce hepatocyte-like cells from human pluripotent stem cells in a 96 well plate format. This process is rapid and cost-effective, allowing the production of quality assured batches of hepatocyte-like cells for basic and applied human research.

• Immunology and Infection

  
  Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae Helen Weavers^1,2, Anna Franz¹, Will Wood³, Paul Martin^{1,4 1}School of Biochemistry, Biomedical Sciences, University of Bristol, ²School of Cellular and Molecular Medicine, Biomedical Sciences, University of Bristol, ³MRC Centre for Inflammation Research, University of Edinburgh, Queens Medical Research Institute, ⁴School of Physiology, Pharmacology, and Neuroscience, Biomedical Sciences, University of Bristol Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

• Immunology and Infection

  
  Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α Alba Llibre*^1,2, Vincent Bondet*^1,2, Mathieu P. Rodero³, David Hunt⁴, Yanick J. Crow^3,5, Darragh Duffy^{1,2 1}Immunobiology of Dendritic Cells, Institut Pasteur, ²INSERM U1223, ³Laboratory of Neurogenetics and Neuroinflammation, INSERM UMR1163, Institut Imagine, ⁴MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, ⁵Manchester Centre for Genomic Medicine, University of Manchester Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-α subtypes in human samples.

• Biology

  
  Oral Bacterial Infection and Shedding in Drosophila melanogaster Jonathon A. Siva-Jothy¹, Arun Prakash¹, Radhakrishnan B. Vasanthakrishnan², Katy M. Monteith¹, Pedro F. Vale^{1,3 1}Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, ²IGDR - CNRS UMR 6290, ³Centre for Immunity, Infection and Evolution, University of Edinburgh This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

• Engineering

  
  Experimental Procedure for Laboratory Studies of In Situ Burning : Flammability and Burning Efficiency of Crude Oil Laurens van Gelderen¹, Grunde Jomaas^{1,2 1}Department of Civil Engineering, Technical University of Denmark, ²School of Engineering, BRE Centre for Fire Safety Engineering, University of Edinburgh Here, we present a protocol to simultaneously study the flammability and burning efficiency of fresh and weathered crude oil under conditions that simulate in situ burning operations on the sea.

• Neuroscience

  
  Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains Julie Mazzolini¹, Kelda Chia¹, Dirk Sieger^{1 1}Centre for Discovery Brain Sciences, University of Edinburgh We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

• Immunology and Infection

  
  Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy Bethany Mills¹, Ahsan R. Akram¹, Emma Scholefield¹, Mark Bradley², Kevin Dhaliwal^{1 1}EPSRC Proteus Hub, MRC Centre of Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, ²School of Chemistry, EaStChem, University of Edinburgh This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

• Developmental Biology

  
  Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification Cui Lin¹, Dongxing Zhu², Greg Markby¹, Brendan M. Corcoran³, Colin Farquharson¹, Vicky E. Macrae^{1 1}Developmental Biology, The Roslin Institute and R(D)SVS, University of Edinburgh, ²Guangzhou Institute of Cardiovascular Disease, The Second Affiliated Hospital, School of Basic Medical Sciences, Guangzhou Medical University, ³Clinical Sciences and R(D)SVS, University of Edinburgh This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

• Developmental Biology

  
  Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells Yu Wang¹, Sharmin Alhaque¹, Kate Cameron¹, Jose Meseguer-Ripolles¹, Baltasar Lucendo-Villarin¹, Hassan Rashidi¹, David C. Hay^{1 1}MRC Centre for Regenerative Medicine, University of Edinburgh The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

• Developmental Biology

  
  Analysis of Coronary Vessels in Cleared Embryonic Hearts Sarah Ivins¹, Catherine Roberts¹, Bertrand Vernay², Peter J. Scambler^{1 1}Developmental Biology of Birth Defects, UCL Institute of Child Health, ²MRC Centre for Regenerative Medicine, SCRM Building, University of Edinburgh We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

• Developmental Biology

  
  Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification Dean A. Houston*¹, Katherine A. Staines*¹, Vicky E. MacRae¹, Colin Farquharson^{1 1}Developmental Biology, The Roslin Institute and R(D)SVS, The University of Edinburgh We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

• Immunology and Infection

  
  A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release Unity Jeffery¹, Robert D. Gray², Dana N. LeVine^{3 1}Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine, Iowa State University, ²MRC Centre for Inflammation Research, University of Edinburgh, ³Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

• Bioengineering

  
  High-throughput Identification of Bacteria Repellent Polymers for Medical Devices Seshasailam Venkateswaran*¹, Peter J. Gwynne*², Mei Wu¹, Ailsa Hardman², Annamaria Lilienkampf¹, Salvatore Pernagallo¹, Garry Blakely², David G. Swann³, Mark Bradley¹, Maurice P. Gallagher^{2 1}School of Chemistry, EaStCHEM, University of Edinburgh, ²School of Biological Sciences, University of Edinburgh, ³Critical Care, NHS Lothian, Royal Infirmary of Edinburgh A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

• Developmental Biology

  
  Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue James E. Baily¹, William C.W. Chen^2,3, Nusrat Khan⁴, Iain R. Murray⁴, Zaniah N. González Galofre⁴, Johnny Huard^5,6, Bruno Péault^{4,7 1}Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, ²Department of Bioengineering and Orthopaedic Surgery, University of Pittsburgh, ³Research Laboratory of Electronics and Department of Biological Engineering, Massachusetts Institute of Technology, ⁴MRC Centre for Regenerative Medicine, University of Edinburgh, ⁵Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh, ⁶Department of Orthopaedic Surgery, University of Texas Health Science Center at Houston, ⁷Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital, David Geffen School of Medicine, University of California at Los Angeles Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146⁺CD34^- pericytes and CD34⁺CD146^- adventitial cells, from the human myocardium.

• Cancer Research

  
  Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window Carolina Rodriguez-Tirado¹, Takanori Kitamura⁵, Yu Kato^1,2, Jeffery W. Pollard^1,2,5, John S. Condeelis^3,4, David Entenberg^{3,4 1}Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, ²Department of Obstetrics/Gynecology and Woman’s Health, Albert Einstein College of Medicine, ³Department of Anatomy & Structural Biology, Albert Einstein College of Medicine, ⁴Gruss-Lipper Biophotonics Center Integrated Imaging Program, Albert Einstein College of Medicine, ⁵Medical Research Council Centre for Reproductive Health, Queen’s Medical Research Institute, University of Edinburgh This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

• Biology

  
  Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter Louise L. Hansen¹, Gerben van Ooijen^{1 1}Institute for Molecular Plant Sciences, University of Edinburgh The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

• Neuroscience

  
  Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction Mario Sanhueza*¹, Anisha Kubasik-Thayil*², Giuseppa Pennetta^{1 1}Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, ²School of Biomedical Sciences, University of Edinburgh Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

• Chemistry

  
  An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions Mohameedyaseen Syedbasha¹, Janina Linnik^1,2,3, Deanna Santer⁴, Daire O'Shea⁵, Khaled Barakat^4,6, Michael Joyce⁴, Nina Khanna⁷, D. Lorne Tyrrell⁴, Michael Houghton⁴, Adrian Egli^{1,8 1}Applied Microbiology Research, Department of Biomedicine, University of Basel, ²Department of Biosystems Science and Engineering, ETH Zurich, and Swiss Institute of Bioinformatics, ³Swiss Institute of Bioinformatics, ⁴Li Ka Shing Institute for Virology, University of Alberta, ⁵Regional Infectious Diseases Unit, University of Edinburgh, ⁶Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, ⁷Infection Biology, Department of Biomedicine, University of Basel, ⁸Clinical Microbiology, University Hospital Basel The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

• Developmental Biology

  
  Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos Yali Huang¹, Ron Wilkie¹, Valerie Wilson^{1 1}MRC Centre for Regenerative Medicine, School of Biological Sciences, University of Edinburgh We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

• Immunology and Infection

  
  Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products Chris J. C. Johnston¹, Elaine Robertson¹, Yvonne Harcus¹, John R. Grainger², Gillian Coakley¹, Danielle J. Smyth¹, Henry J. McSorley¹, Rick Maizels^{1 1}Institute of Immunology and Infection Research, University of Edinburgh, ²Manchester Collaborative Centre for Inflammation Research Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

• Developmental Biology

  
  Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles Stephanie Morgan¹, Lisa Campbell¹, Vivian Allison¹, Alison Murray², Norah Spears^{1 1}Centre for Integrative Physiology, University of Edinburgh, ²MRC Centre for Reproductive Health, University of Edinburgh This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

• Developmental Biology

  
  Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin Thomas Ramezani¹, Derek W. Laux¹, Isabel R. Bravo¹, Masazumi Tada², Yi Feng^{1 1}MRC Centre for Inflammation Research, Queen's Medical Research Institute, The University of Edinburgh, ²Department of Cell & Developmental Biology, University College London Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRAS^G12V expressing skin cells in zebrafish larvae.

• Medicine

  
  A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction Emily E. Hesketh¹, Madeleine A. Vernon¹, Peng Ding¹, Spike Clay¹, Gary Borthwick¹, Bryan Conway¹, Jeremy Hughes^{1 1}MRC Centre for Inflammation Research, University of Edinburgh The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

• Biology

  
  A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies Samantha L. Eaton¹, Maica Llavero Hurtado¹, Karla J. Oldknow², Laura C. Graham¹, Thomas W. Marchant¹, Thomas H. Gillingwater^3,4, Colin Farquharson², Thomas M. Wishart^{1,4 1}Division of Neurobiology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, ²Division of Developmental Biology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, ³Centre for Integrative Physiology, University of Edinburgh, ⁴Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

• Neuroscience

  
  Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey Antón Barreiro-Iglesias¹, Guixin Zhang², Michael E. Selzer^2,3, Michael I. Shifman^{2 1}Centre for Neuroregeneration, School of Biomedical Sciences, University of Edinburgh, ²Shriners Hospitals Pediatric Research Center (Center for Neural Repair and Rehabilitation), Temple University School of Medicine, ³Department of Neurology, Temple University School of Medicine Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

• Medicine

  
  Mouse Kidney Transplantation: Models of Allograft Rejection George H. Tse*¹, Emily E. Hesketh*¹, Michael Clay¹, Gary Borthwick¹, Jeremy Hughes¹, Lorna P. Marson^{1 1}MRC Centre for Inflammation Research, The Queen's Medical Research Institute, The University of Edinburgh Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

• Chemistry

  
  Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces Baltasar Lucendo-Villarin¹, Kate Cameron¹, Dagmara Szkolnicka¹, Paul Travers¹, Ferdous Khan², Jeffrey G. Walton², John Iredale³, Mark Bradley², David C. Hay^{1 1}MRC Centre for Regenerative Medicine, University of Edinburgh, ²School of Chemistry, University of Edinburgh, ³MRC Centre for Inflammation Research, University of Edinburgh This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human hepatocytes.

• Medicine

  
  Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration Emily E. Hesketh¹, Alicja Czopek¹, Michael Clay¹, Gary Borthwick¹, David Ferenbach¹, David Kluth¹, Jeremy Hughes^{1 1}MRC Centre for Inflammation Research, University of Edinburgh The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

• Biology

  
  Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration Richard L. Mort¹, Margaret Keighren¹, Leonard Hay¹, Ian J. Jackson^{1 1}MRC Human Genetics Unit, MRC IGMM, Western General Hospital, University of Edinburgh We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

• Bioengineering

  
  Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates Mark A. Hughes¹, Paul M. Brennan², Andrew S. Bunting³, Mike J. Shipston¹, Alan F. Murray^{3 1}Centre for Integrative Physiology, School of Biomedical Sciences, The University of Edinburgh, ²Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, Western General Hospital, ³School of Engineering, Institute for Integrated Micro and Nano Systems, The University of Edinburgh This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

• Neuroscience

  
  Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis Lyndsay Murray¹, Thomas H Gillingwater², Rashmi Kothary^{1 1}Regenerative Medicine Program, Ottawa Hospital Research Institute, ²Centre for Integrative Physiology, University of Edinburgh In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

• Immunology and Infection

  
  Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin Cecile Benezech^1,2, Jorge H. Caamano^{1 1}School of Immunity and Infection, IBR-MRC Centre for Immune Regulation, College of Medical and Dental Sciences, University of Birmingham, ²Centre for Cardiovascular Sciences, University of Edinburgh Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

• Engineering

  
  Sputter Growth and Characterization of Metamagnetic B2-ordered FeRh Epilayers Chantal Le Graët¹, Mark A. de Vries^1,2, Mathew McLaren^1,3, Richard M.D. Brydson³, Melissa Loving⁴, Don Heiman⁵, Laura H. Lewis⁴, Christopher H. Marrows^{1 1}School of Physics and Astronomy, University of Leeds, ²Institute of Materials Research, University of Leeds, ³School of Chemistry, University of Edinburgh, ⁴Department of Chemical Engineering, Northeastern University, ⁵Department of Physics, Northeastern University A method to prepare epitaxial layers of ordered alloys by sputtering is described. The B2-ordered FeRh compound is used as an example, as it displays a metamagnetic transition that depends sensitively on the degree of chemical order and the exact composition of the alloy.

• Medicine

  
  The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers Fiach C. O'Mahony¹, Jyoti Nanda¹, Alexander Laird¹, Peter Mullen², Helen Caldwell³, Ian M. Overton⁴, Lel Eory⁴, Marie O'Donnell^1,5, Dana Faratian⁶, Thomas Powles⁷, David J. Harrison^1,2, Grant D. Stewart^{1 1}Edinburgh Urological Cancer Group, University of Edinburgh, ²School of Medicine, University of St Andrews, ³Division of Pathology, University of Edinburgh, ⁴MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, ⁵Department of Pathology, Western General Hospital, ⁶Breakthrough Breast Cancer Research Unit, University of Edinburgh, ⁷St Bartholomew's Cancer Institute, Experimental Cancer Medicine Centre, Queen Mary University of London RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

• Biology

  
  Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH) Elena Ronander^1,2, Dominique C. Bengtsson^1,2, Louise Joergensen^1,2, Anja T. R. Jensen^1,2, David E. Arnot^{1,2,3 1}Centre for Medical Parasitology, Department of International Health, Immunology & Microbiology, Faculty of Health Sciences, University of Copenhagen, ²Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), ³Institute of Infection and Immunology Research, School of Biology, University of Edinburgh Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes ¹. The technique is adaptable and can be used on different types of genes, cells and organisms.

• Biology

  
  Genomic Transformation of the Picoeukaryote Ostreococcus tauri Gerben van Ooijen¹, Kirsten Knox¹, Katalin Kis¹, François-Yves Bouget^2,3, Andrew J. Millar^{1 1}SynthSys, University of Edinburgh, ²Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, Paris 06, ³UMR 7621, Laboratoire d'Océanographie Microbienne, Observatoire Océanologique, Banyuls-sur-Mer, Université Pierre et Marie Curie, Paris 06 This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

• Bioengineering

  
  Use of Human Perivascular Stem Cells for Bone Regeneration Aaron W. James*¹, Janette N. Zara*², Mirko Corselli², Michael Chiang¹, Wei Yuan², Virginia Nguyen¹, Asal Askarinam¹, Raghav Goyal¹, Ronald K. Siu³, Victoria Scott¹, Min Lee³, Kang Ting¹, Bruno Péault^2,4, Chia Soo^{2 1}Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, UCLA, ²UCLA and Orthopaedic Hospital, Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, UCLA, ³Department of Bioengineering, UCLA, ⁴Center for Cardiovascular Science, University of Edinburgh Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

• Neuroscience

  
  Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex Hugh Pastoll¹, Melanie White², Matthew Nolan^{2 1}Neuroinformatics DTC, University of Edinburgh, ²Centre for Integrative Physiology, University of Edinburgh We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

• Immunology and Infection

  
  Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells Antoine Claessens¹, J. Alexandra Rowe^{1 1}Centre for Immunity, Infection and Evolution, University of Edinburgh An in vitro model for cerebral malaria sequestration is described¹. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

• Neuroscience

  
  Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes Giselle Cheung¹, Michael A. Cousin^{1 1}Membrane Biology Group, Centre for integrative Physiology, University of Edinburgh A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

• Biology

  
  Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations Claire N. Medine¹, Baltasar Lucendo-Villarin¹, Wenli Zhou¹, Christopher C. West¹, David C. Hay^{1 1}Medical Research Council Centre for Regenerative Medicine, University of Edinburgh This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

• Medicine

  
  Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence Dana Faratian¹, Jason Christiansen², Mark Gustavson², Christine Jones², Christopher Scott², InHwa Um¹, David J. Harrison^{1 1}Division of Pathology, University of Edinburgh, ²HistoRx Inc. Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

• Biology

  
  Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography Alexander D. Leeper¹, Joanne Farrell², J. Michael Dixon¹, Sarah E. Wedden², David J. Harrison¹, Elad Katz^{1 1}Breakthrough Breast Cancer Research Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, ²MRC Technology We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.