University of Edinburgh View Institution's Website 58 articles published in JoVE Developmental Biology In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology Telma Ventura1, Ewan Joshua Egan1, Nicola Romanò2, Antonella Fidanza1 1Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh, 2Centre for Discovery Brain Sciences, University of Edinburgh Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation. 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 Cazier2,3, 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 Nonino6 1Department 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. Hay1 1MRC 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. Hay1 1MRC 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 Granneman1 1Centre 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-Yrigoyen1,2, Alisha May1, Telma Ventura1, Helen Taylor1, Antonella Fidanza1, Luca Cassetta2, Jeffrey W. Pollard2, Lesley M. Forrester1 1Centre 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 Park3 1School 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 Wang2,3, Alistair J. McCormick1,2 1Institute 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 Vezzani1,2, Mario Gomez-Salazar1, Joan Casamitjana1, Carlo Tremolada3, Bruno Péault1,4 1MRC 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. Beggs1 1Wellcome 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 Rashidi1,2, Sharmin Alhaque1,3, Lena Fischer1,4, Jose Meseguer-Ripolles1, Yu Wang1, Cliona O'Farrelly4, Michael Themis3, David C. Hay1 1MRC 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-Ochoa1,2, Isabella E. Maudlin1,3, Emanuela Sani1, Jean D. Beggs1 1Wellcome 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 Blin1 1MRC 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 Huang1,2, Dinja van der Hoorn*1,2, Leire M. Ledahawsky*1,2, Anna A. L. Motyl*1,2, Crispin Y. Jordan1, Thomas H. Gillingwater1,2, Ewout J. N. Groen1,2 1Centre 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. Carragher3,4 1Royal (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. Hay1 1MRC 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 Weavers1,2, Anna Franz1, Will Wood3, Paul Martin1,4 1School 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. Crow3,5, Darragh Duffy1,2 1Immunobiology 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. Vale1,3 1Institute 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 Jomaas1,2 1Department 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 Sieger1 1Centre 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 Dhaliwal1 1EPSRC 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. Macrae1 1Developmental 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. Hay1 1MRC 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. Scambler1 1Developmental 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 Farquharson1 1Developmental 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. LeVine3 1Department 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. Gallagher2 1School 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. Chen2,3, Nusrat Khan4, Iain R. Murray4, Zaniah N. González Galofre4, Johnny Huard5,6, Bruno Péault4,7 1Centre 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 Kato1,2, Jeffery W. Pollard1,2,5, John S. Condeelis3,4, David Entenberg3,4 1Department 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 Ooijen1 1Institute 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 Pennetta1 1Euan 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 Linnik1,2,3, Deanna Santer4, Daire O'Shea5, Khaled Barakat4,6, Michael Joyce4, Nina Khanna7, D. Lorne Tyrrell4, Michael Houghton4, Adrian Egli1,8 1Applied 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 Wilson1 1MRC 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 Maizels1 1Institute 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 Spears1 1Centre 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 Feng1 1MRC 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 HRASG12V 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 Hughes1 1MRC 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. Gillingwater3,4, Colin Farquharson2, Thomas M. Wishart1,4 1Division 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. Selzer2,3, Michael I. Shifman2 1Centre 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. Marson1 1MRC 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. Hay1 1MRC 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 Hughes1 1MRC 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. Jackson1 1MRC 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. Murray3 1Centre 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 Kothary1 1Regenerative 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 Benezech1,2, Jorge H. Caamano1 1School 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 Vries1,2, Mathew McLaren1,3, Richard M.D. Brydson3, Melissa Loving4, Don Heiman5, Laura H. Lewis4, Christopher H. Marrows1 1School 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'Donnell1,5, Dana Faratian6, Thomas Powles7, David J. Harrison1,2, Grant D. Stewart1 1Edinburgh 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 Ronander1,2, Dominique C. Bengtsson1,2, Louise Joergensen1,2, Anja T. R. Jensen1,2, David E. Arnot1,2,3 1Centre 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 Bouget2,3, Andrew J. Millar1 1SynthSys, 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 Péault2,4, Chia Soo2 1Dental 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 Nolan2 1Neuroinformatics 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 Rowe1 1Centre 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. Cousin1 1Membrane 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. Hay1 1Medical 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. Harrison1 1Division 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 Katz1 1Breakthrough 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.