University of Oxford

57 articles published in JoVE

• Biology

  
  Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging Yuewen Sheng¹, Kyle Morris¹, Julika Radecke*¹, Peijun Zhang*^{1,2,3 1}Electron Bio-Imaging Centre, Diamond Light Source Ltd, Harwell Science & Innovation Campus, ²Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, ³Chinese Academy of Medical Sciences Oxford Institute, University of Oxford The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 Å resolution.

• Immunology and Infection

  
  Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology Marina Diotallevi*^1,2, Thomas Nicol*^1,2, Faseeha Ayaz^1,2, Jade Bailey^1,2, Andrew Shaw^1,2, Eileen McNeill^1,2, Ben Davies^1,2, Keith M. Channon^1,2, Mark J. Crabtree^{1,2 1}BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, ²Wellcome Centre for Human Genetics, University of Oxford This protocol has been established to culture tetrahydrobiopterin (BH4)- and inducible nitric oxide synthase (iNOS)-deficient primary murine macrophages to study NO-redox biology. The study focuses on reducing potential contamination of BH4 and other artifacts found in traditional isolation and culture methods which may confound experimental outcomes and interpretation of results.

• Immunology and Infection

  
  Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses Pablo F. Céspedes¹, Michael L. Dustin^{1 1}Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, The University of Oxford Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

• Engineering

  
  UV-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media Ana C. Quevedo¹, Emily Guggenheim¹, Sophie M. Briffa¹, Jessica Adams^2,3, Stephen Lofts², Minjeong Kwak⁴, Tae Geol Lee⁴, Colin Johnston⁵, Stephan Wagner⁶, Timothy R. Holbrook⁶, Yves U. Hachenberger⁷, Jutta Tentschert⁷, Nicholas Davidson¹, Eugenia Valsami-Jones^{1 1}School of Geography, Earth and Environmental Sciences, University of Birmingham, ²UK Centre for Ecology and Hydrology, ³Natural England, ⁴Center for Nanosafety Metrology, Korea Research Institute of Standards and Science (KRISS), ⁵Department of Materials, University of Oxford, ⁶Department of Analytical Chemistry, Helmholtz-Centre for Environmental Research, ⁷Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR) This study presents the benchmarking results for an interlaboratory comparison (ILC) designed to test the standard operating procedure (SOP) developed for gold (Au) colloid dispersions characterized by ultraviolet-visible Spectroscopy (UV-Vis), amongst six partners from the H2020 ACEnano project for sample preparation, measurement, and analysis of the results.

• Biology

  
  Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells Nina Vyas*¹, Nina Perry*¹, Chidinma A. Okolo¹, Ilias Kounatidis¹, Thomas M. Fish¹, Kamal L. Nahas^1,2, Archana Jadhav¹, Mohamed A. Koronfel¹, Johannes Groen³, Eva Pereiro³, Ian M. Dobbie⁴, Maria Harkiolaki^{1 1}Harwell Science and Innovation Campus, Beamline B24, Diamond Light Source, ²Division of Virology, Department of Pathology, University of Cambridge, ³ALBA Synchrotron, Beamline 09 - MISTRAL, ⁴Micron Advanced Imaging Consortium, Department of Biochemistry, University of Oxford This protocol demonstrates how to image biological cryo-preserved samples using cryo-structured illumination microscopy. We demonstrate the methodology by imaging the cytoskeleton of U2OS cells.

• Bioengineering

  
  Studying Cavitation Enhanced Therapy Michael Gray*¹, Alexandra V. Vasilyeva*¹, Veerle Brans*¹, Eleanor Stride^{1 1}Institute of Biomedical Engineering, University of Oxford The presented experimental protocol can be used to perform real time measurements of cavitation activity in a cell culture device with the aim of enabling investigation of the conditions required for successful drug delivery and/or other bioeffects.

• Neuroscience

  
  In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages Hazel Hall-Roberts^1,2,3, Elena Di Daniel², William S. James¹, John B. Davis², Sally A. Cowley^{1 1}James Martin Stem Cell Facility, Sir William Dunn School of Pathology, University of Oxford, ²Alzheimer’s Research UK Oxford Drug Discovery Institute, Nuffield Department of Medicine Research Building, University of Oxford, ³UK Dementia Research Institute, Cardiff University Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

• Chemistry

  
  Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison Sophie M. Briffa¹, Jo Sullivan², Agnieszka Siupa², Pauline Carnell-Morris², Michele Carboni², Kerstin Jurkschat³, Ruud J. B. Peters⁴, Carolin Schultz⁵, Kang Hee Seol⁶, Sook-Jin Kwon⁶, Sehee Park⁷, Tae Hyun Yoon^6,7, Colin Johnston³, Stephen Lofts⁸, Eugenia Valsami-Jones^{1 1}School of Geography, Earth and Environmental Sciences, University of Birmingham, ²Malvern Panalytical, ³Department of Materials, University of Oxford, ⁴Wageningen Food Safety Research, ⁵UK Centre for Ecology & Hydrology, ⁶Institute for Next Generation Material Design, Hanyang University, ⁷Department of Chemistry, College of Natural Sciences, Hanyang University, ⁸UK Centre for Ecology & Hydrology The protocol described here aims to measure the hydrodynamic diameter of spherical nanoparticles, more specifically gold nanoparticles, in aqueous media by means of Nanoparticle Tracking Analysis (NTA). The latter involves tracking the movement of particles due to Brownian motion and implementing the Stokes-Einstein equation to obtain the hydrodynamic diameter.

• Biology

  
  High-Accuracy Correction of 3D Chromatic Shifts in the Age of Super-Resolution Biological Imaging Using Chromagnon Atsushi Matsuda^1,2, Takako Koujin¹, Lothar Schermelleh³, Tokuko Haraguchi^1,2, Yasushi Hiraoka^{1,2 1}Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology, ²Graduate School of Frontier Biosciences, Osaka University, ³Micron Advanced Bioimaging Unit, Department of Biochemistry, University of Oxford Correction of chromatic shifts in three-dimensional (3D) multicolor fluorescence microscopy images is crucial for quantitative data analyses. This protocol is developed to measure and correct chromatic shifts in biological samples through acquisition of suitable reference images and processing with the open-source software, Chromagnon.

• Developmental Biology

  
  Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish Tomasz Dobrzycki*^1,2, Monika Krecsmarik*^1,2, Rui Monteiro^{1,2,3 1}MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, ²BHF Centre of Research Excellence, ³Institute of Cancer and Genomic Sciences, University of Birmingham Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

• Biology

  
  Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells Alexander Hamilton^1,2, Elisa Vergari¹, Caroline Miranda³, Andrei I. Tarasov^{1,4,5 1}Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, ²Lund University Diabetes Centre, Unit of Molecular Metabolism, Clinical Research Centre, Malmö University HospitalMalmö, ³Institute of Neuroscience of Physiology, Department of Physiology, Metabolic Research Unit, University of Göteborg, ⁴Oxford National Institute for Health Research, Biomedical Research Centre, ⁵Life and Medical Sciences, University of Hertfordshire Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

• Behavior

  
  Investigating the Effect of Visual Imagery and Learning Shape-Audio Regularities on Bouba and Kiki Torø Graven¹, Clea Desebrock^{1 1}Department of Experimental Psychology, University of Oxford The aim of the presented protocol is to investigate the role of visual imagery in the bouba/kiki-effect, whether training in noticing the bouba/kiki shape-audio regularities affects the bouba/kiki-effect and the recognition of individual bouba and kiki shapes, and finally what mental images these regularities produce.

• Editorial

  
  Chloroplast Research Methods: Probing The Targeting, Localization And Interactions Of Chloroplast Proteins Paul Jarvis^{1 1}Department of Plant Sciences, University of Oxford

• 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.

• Immunology and Infection

  
  Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection Julie M. Mazet¹, Ana L. Chiodetti¹, Jagdish N. Mahale¹, Audrey Gérard^{1 1}The Kennedy Institute of Rheumatology, The University of Oxford Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

• Behavior

  
  An Instrumented Pull Test to Characterize Postural Responses Joy Tan^1,2,4, Wesley Thevathasan^2,3,4,5, Jennifer McGinley⁶, Peter Brown⁷, Thushara Perera^{1,4 1}Department of Medical Bionics, The University of Melbourne, ²Department of Neurology, The Royal Melbourne Hospital, ³Department of Neurology, Austin Hospital, ⁴The Bionics Institute, ⁵Department of Medicine, The University of Melbourne, ⁶Department of Physiotherapy, The University of Melbourne, ⁷Medical Research Council Brain Network Dynamics Unit, University of Oxford Impairment of postural reflexes, termed postural instability, is difficult to quantify. Clinical assessments such as the pull test suffer issues with reliability and scaling. Here, we present an instrumented version of the pull test to objectively characterize postural responses.

• Biochemistry

  
  Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis Alicia Ventresca Miller¹, Ricardo Fernandes^1,2, Anneke Janzen¹, Ayushi Nayak¹, Jillian Swift¹, Jana Zech¹, Nicole Boivin¹, Patrick Roberts^{1 1}Max Planck Institute for the Science of Human History, ²School of Archaeology, University of Oxford Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

• Genetics

  
  Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents Naemeh Pourshafie¹, Philip R. Lee², Ke-lian Chen¹, George G. Harmison¹, Laura C. Bott^1,3, Kenneth H. Fischbeck¹, Carlo Rinaldi^{1,4 1}Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, ²Section on Nervous System Development and Plasticity, The Eunice Kennedy Shriver National Institute of Child and Human Development, National Institutes of Health, ³Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University, ⁴Department of Physiology, Anatomy and Genetics, University of Oxford Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

• Biochemistry

  
  Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software Rory Nolan¹, Luis A. Alvarez¹, Samuel C. Griffiths², Jonathan Elegheert², Christian Siebold², Sergi Padilla-Parra^{1,2,3,4 1}Cellular Imaging Group, Wellcome Centre Human Genetics, University of Oxford, ²Division of Structural Biology, Wellcome Centre Human Genetics, University of Oxford, ³Dynamic Structural Virology Group, Biocruces Health Research Centre, ⁴IKERBASQUE, Basque Foundation for Science This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

• Immunology and Infection

  
  Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins Meisam Naeimi Kararoudi¹, Hamid Dolatshad², Prashant Trikha¹, Syed-Rehan A. Hussain³, Ezgi Elmas¹, Jennifer A. Foltz¹, Jena E. Moseman¹, Aarohi Thakkar¹, Robin J. Nakkula¹, Margaret Lamb¹, Nitin Chakravarti¹, K. John McLaughlin³, Dean A. Lee^{1 1} Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor–b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

• Genetics

  
  Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance Jose Antonio Escudero^1,2, R Craig MacLean³, Alvaro San Millan^{4 1}Department of Animal Health, Universidad Complutense de Madrid, ²Visavet Health Surveillance Centre, Universidad Complutense de Madrid, ³Department of Zoology, University of Oxford, ⁴Department of Microbiology, Hospital Universitario Ramon y Cajal (IRYCIS) and CIBERESP Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

• Biochemistry

  
  Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes. Mikhail A. Galkin¹, Aidan N. Russell², Steven B. Vik³, Richard M. Berry², Robert R. Ishmukhametov^{2 1}First Pavlov State Medical University, ²Clarendon Laboratory, Department of Physics, Oxford University, ³Department of Biological Sciences, Southern Methodist University Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

• Neuroscience

  
  Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol Anne C. Wolfes^1,2,3, Camin Dean^{3 1}Chemical Biology, Chemistry Research Laboratory, University of Oxford, ²Department of Physiology, Anatomy, and Genetics, University of Oxford, ³Trans-synaptic signaling, European Neuroscience Institute The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca²⁺ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

• Biochemistry

  
  Protein Film Infrared Electrochemistry Demonstrated for Study of H₂ Oxidation by a [NiFe] Hydrogenase Philip A. Ash*¹, Ricardo Hidalgo*¹, Kylie A. Vincent^{1 1}Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory Here, we describe a technique, protein film infrared electrochemistry, which allows immobilized redox proteins to be studied spectroscopically under direct electrochemical control at a carbon electrode. Infrared spectra of a single protein sample can be recorded at a range of applied potentials and under a variety of solution conditions.

• Medicine

  
  In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice Evandro F. Fang^1,6, Konstantinos Palikaras², Nuo Sun³, Elayne M. Fivenson¹, Ryan D. Spangler⁴, Jesse S. Kerr¹, Stephanie A. Cordonnier¹, Yujun Hou¹, Eszter Dombi⁵, Henok Kassahun⁶, Nektarios Tavernarakis^2,7, Joanna Poulton⁵, Hilde Nilsen⁶, Vilhelm A. Bohr^{1,8 1}Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, ²Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology - Hellas, ³Center for Molecular Medicine, National Heart Lung and Blood Institute, National Institutes of Health, ⁴Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, ⁵Nuffield Department of Obstetrics and Gynaecology, University of Oxford, ⁶Department of Clinical Molecular Biology, University of Oslo and Akershus University Hospital, ⁷Department of Basic Sciences, Faculty of Medicine, University of Crete, ⁸Danish Center for Healthy Aging, University of Copenhagen Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

• Neuroscience

  
  Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation Anna Shpektor^1,3, Maria Nazarova⁴, Matteo Feurra^{1,2 1}School of Psychology, Centre for Cognition and Decision Making, National Research University Higher School of Economics, ²Department of Medicine, Surgery and Neuroscience, Unit of Neurology and Clinical Neurophysiology, Brain Investigation & Neuromodulation Lab. (Si-BIN Lab), Azienda Ospedaliera Universitaria of Siena, ³Department of Experimental Psychology, University of Oxford, ⁴Centre for Cognition and Decision Making, National Research University Higher School of Economics Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

• Chemistry

  
  Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene Rebecca A. Ryan¹, Sophie Williams¹, Andrew V. Martin¹, Ruben A. Dilanian¹, Connie Darmanin², Corey T. Putkunz¹, David Wood³, Victor A. Streltsov⁴, Michael W.M. Jones⁵, Naylyn Gaffney⁶, Felix Hofmann⁷, Garth J. Williams⁸, Sebastien Boutet⁹, Marc Messerschmidt¹⁰, M. Marvin Seibert¹¹, Evan K. Curwood¹¹, Eugeniu Balaur², Andrew G. Peele⁵, Keith A. Nugent², Harry M. Quiney¹, Brian Abbey^{2 1}ARC Centre of Excellence in Advanced Molecular Imaging, School of Physics, University of Melbourne, ²Australian Research Council (ARC) Centre of Excellence in Advanced Molecular Imaging, Department of Chemistry and Physics, La Trobe Institute for Molecular Sciences, La Trobe University, ³Department of Physics, Imperial College London, ⁴Florey Institute of Neuroscience and Mental Health, ⁵Science and Engineering Faculty, Queensland University of Technology, ⁶Swinburne University of Technology, ⁷Department of Engineering Science, University of Oxford, ⁸Brookhaven National Laboratory, ⁹Linac Coherent Light Source, SLAC National Accelerator Laboratory, ¹⁰BioXFEL Science and Technology Center, ¹¹Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, ¹²Australian Synchrotron We describe an experiment designed to probe the electronic damage induced in nanocrystals of Buckminsterfullerene (C60) by intense, femtosecond pulses of X-rays. The experiment found that, surprisingly, rather than being stochastic, the X-ray induced electron dynamics in C60 are highly correlated, extending over hundreds of unit cells within the crystals¹.

• Developmental Biology

  
  A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development Charlotte Kirchhelle¹, Ian Moore^{1 1}Department of Plant Sciences, University of Oxford Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

• Immunology and Infection

  
  Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry Stephen C. Juvet¹, Sajad Moshkelgosha², Sharon Sanderson³, Joanna Hester⁴, Kathryn J. Wood⁴, Andrew Bushell^{4 1}Division of Respirology, Departments of Medicine and Immunology, Toronto Lung Transplant Program, Multiorgan Transplant Program, Toronto General Research Institute, University of Toronto and University Health Network, ²Latner Thoracic Surgery Laboratories, Toronto General Research Institute, University Health Network, ³National Institutes of Health Research, Oxford Biomedical Research Centre, Translational Immunology Laboratory, NDORMS, Kennedy Institute of Rheumatology, University of Oxford, ⁴Transplantation Research Immunology Group, Nuffield Department of Surgical Sciences, John Radcliffe Hospital, University of Oxford This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

• Biology

  
  Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants Qihua Ling¹, Paul Jarvis^{1 1}Department of Plant Sciences, University of Oxford Here we describe a new method to study protein import into isolated chloroplasts under stress. The method is rapid and straightforward, and can be applied to study the consequences of different stress conditions for chloroplast protein import, and the corresponding regulatory mechanisms.

• Immunology and Infection

  
  High-throughput Gene Tagging in Trypanosoma brucei Philip Dyer¹, Samuel Dean¹, Jack Sunter^{1 1}Sir William Dunn School of Pathology, University of Oxford Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

• Medicine

  
  Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function Chrystalina A. Antoniades¹, James J. FitzGerald^{2 1}Nuffield Department of Clinical Neuroscience, The University of Oxford, ²Nuffield Department of Surgical Science, The University of Oxford This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

• Medicine

  
  Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome Alicja M. Baczynska^1,2, Sarah Shaw³, Helen C. Roberts^1,2,3,5, Cyrus Cooper^2,3,4, Avan Aihie Sayer^1,2,3,5,6, Harnish P. Patel^{1,2,3 1}Academic Geriatric Medicine, University of Southampton, University Hospital Southampton, ²National Institute for Health Research Southampton Biomedical Research Center, University of Southampton and University Hospital Southampton NHS Foundation Trust, ³MRC Lifecourse Epidemiology Unit, University of Southampton, ⁴National Institute for Health Research Musculoskeletal Biomedical Research Unit, University of Oxford, ⁵National Institute for Health Research Collaboration for Leadership in Applied Health Research and Care, ⁶Newcastle University Institute of Ageing and Institute of Health and Society, Newcastle University This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

• Medicine

  
  Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability Christina Wayman*^1,2, Denise A. Duricki*^1,2, Lisa A. Roy³, Barbara Haenzi¹, Shi-Yen Tsai⁴, Gwendolyn Kartje^4,5,6, John S. Beech⁷, Diana Cash², Lawrence Moon^{1 1}Department of Neuroimaging, James Black Centre, Institute of Psychiatry, King's College London, University of London, ³Institute of Neuroscience and Psychology, Wellcome Surgical Institute, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, ⁴Research Service, Edward Hines Jr. VA Hospital, ⁵Neurology Service, Edward Hines Jr. VA Hospital, ⁶Department of Molecular Pharmacology and Therapeutics, Neuroscience Research Institute, Loyola University Chicago, ⁷Department of Oncology, The Gray Institute for Radiation, Oncology and Biology, University of Oxford Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

• Biology

  
  Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation Louise Aigrain^1,2, Marko Sustarsic¹, Robert Crawford¹, Anne Plochowietz¹, Achillefs N. Kapanidis^{1 1}Clarendon Laboratory, Department of Physics, University of Oxford, ²Wellcome Trust Sanger Institute, Genome Center Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

• Biology

  
  The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example Brendan M. O'Leary¹, Arantza Rico², Sarah McCraw¹, Helen N. Fones³, Gail M. Preston^{1 1}Department of Plant Sciences, University of Oxford, ²School of Education of Vitoria-Gasteiz, University of the Basque Country (UPV/EHU), ³Biosciences, University of Exeter This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

• Immunology and Infection

  
  Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo Juha T. Huiskonen¹, Marie-Laure Parsy¹, Sai Li¹, David Bitto¹, Max Renner¹, Thomas A. Bowden^{1 1}Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

• Biology

  
  Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions Carina Monico^1,2, Gionata Belcastro¹, Francesco Vanzi^1,3, Francesco S. Pavone^1,4,5,6, Marco Capitanio^{1,4 1}LENS - European Laboratory for Non-linear Spectroscopy, University of Florence, ²Chemistry Research Laboratory, University of Oxford, ³Department of Biology, University of Florence, ⁴Department of Physics and Astronomy, University of Florence, ⁵National Institute of Optics-National Research Council, Italy, ⁶International Center of Computational Neurophotonics Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

• Medicine

  
  Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation Simon Little¹, Alek Pogosyan¹, Spencer Neal², Ludvic Zrinzo², Marwan Hariz², Thomas Foltynie², Patricia Limousin², Peter Brown^{1 1}Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, ²Sobell Department of Motor Neuroscience & Movement Disorders, Unit of Functional Neurosurgery, UCL Institute of Neurology Adaptive deep brain stimulation (aDBS) is effective for Parkinson’s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of stimulation.

• Behavior

  
  Stimulating the Lip Motor Cortex with Transcranial Magnetic Stimulation Riikka Möttönen¹, Jack Rogers¹, Kate E. Watkins^{1 1}Department of Experimental Psychology, University of Oxford Transcranial magnetic stimulation (TMS) has proven to be a useful tool in investigating the role of the articulatory motor cortex in speech perception.  This article describes how to record motor evoked potentials (MEPs) from the lip muscles and how to disrupt the motor lip representation using repetitive TMS.

• Biology

  
  Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies Luke A. Yates¹, Robert J. C. Gilbert^{1 1}Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

• Immunology and Infection

  
  Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking Stephan Uphoff^1,2, David J. Sherratt¹, Achillefs N. Kapanidis^{2 1}Microbiology Unit, Department of Biochemistry, University of Oxford, ²Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

• Environment

  
  Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil Danilo O. Carvalho^1,2, Derric Nimmo¹, Neil Naish¹, Andrew R. McKemey¹, Pam Gray¹, André B. B. Wilke³, Mauro T. Marrelli³, Jair F. Virginio⁴, Luke Alphey^1,5, Margareth L. Capurro^{2,6 1}Oxitec Ltd, ²Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, ³Departamento de Epidemiologia, Universidade de São Paulo, ⁴Moscamed Brasil, ⁵Deptartment of Zoology, University of Oxford, ⁶Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM) To achieve population suppression of Aedes aegypti using the RIDL^® (Release of Insects carrying a Dominant Lethal) system, large numbers of male mosquitoes need to be released. This requires the use of mass rearing techniques and technology to provide reliable systems to obtain the maximum number of high quality male mosquitoes.

• Biology

  
  A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases Penelope A. Mason¹, Ivan Boubriak¹, Lynne S. Cox^{1 1}Department of Biochemistry, University of Oxford Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

• Behavior

  
  The Successive Alleys Test of Anxiety in Mice and Rats Robert M.J. Deacon^{1 1}Department of Experimental Psychology, University of Oxford The plus-maze measures anxiety-like behaviour in rodents. There are two opposite closed and two opposite open arms; anxious rodents avoid the open arms. The central area is neither completely open nor closed, so time spent here is ambiguous and difficult to interpret. Here a modification of the plus-maze protocol eliminating this area is described.

• Behavior

  
  Shallow Water (Paddling) Variants of Water Maze Tests in Mice Robert M.J. Deacon^{1 1}Department of Experimental Psychology, University of Oxford Mice can swim, but many strains appear to find this activity stressful. To overcome this problem mazes have been devised where escape from shallow water is used to motivate behaviour. These have been demonstrated to support learning at least as good as the traditional and widely used Morris water maze.

• Medicine

  
  Measuring the Strength of Mice Robert M.J. Deacon^{1 1}Department of Experimental Psychology, University of Oxford Deficits in muscular strength occur in many clinical conditions such as motor neuron disease. The inverted screen and weight lifting tests described here measure strength in mice almost exclusively, with minimal influence of factors such as coordination.

• Neuroscience

  
  Measuring Motor Coordination in Mice Robert M.J. Deacon^{1 1}Department of Experimental Psychology, University of Oxford Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

• Biology

  
  Preparing Individual Drosophila Egg Chambers for Live Imaging Timothy T. Weil¹, Richard M. Parton¹, Ilan Davis^{1 1}Department of Biochemistry, University of Oxford The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

• Neuroscience

  
  Assessing Burrowing, Nest Construction, and Hoarding in Mice Robert Deacon^{1 1}Department of Experimental Psychology, University of Oxford Burrowing, nesting, and hoarding are species-typical activities that mice readily perform in the laboratory. This article describes how they can be easily and cheaply assessed. These protocols are extremely sensitive to mouse strain, brain lesions and diseases. Moreover they constitute “environmental enrichment” for the mice, and embody the “Refinement” aspect of the “3 Rs”18.

• Neuroscience

  
  Electrophysiological Measurements and Analysis of Nociception in Human Infants L. Fabrizi*¹, A. Worley*², D. Patten¹, S. Holdridge¹, L. Cornelissen¹, J. Meek³, S. Boyd², R. Slater^{1,4 1}Neuroscience, Physiology and Pharmacology, University College London, ²Department of Clinical Neurophysiology, Great Ormond Street Hospital, ³Elizabeth Garrett Anderson Obstetric Hospital, University College Hospital, ⁴Nuffield Department of Anaesthetics, University of Oxford The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

• Biology

  
  Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique Rebekka A.V. Schwab¹, Wojciech Niedzwiedz^{1,2 1}Department of Molecular Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, ²Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

• Neuroscience

  
  Hyponeophagia: A Measure of Anxiety in the Mouse Rob M.J. Deacon^{1 1}Department of Experimental Psychology, University of Oxford Mice and rats, due to their innate cautiousness, are initially slow in consuming a novel food, particularly in a novel place. This hyponeophagia can readily be measured in the laboratory, even though laboratory animals are much less anxious than their wild counterparts

• Biology

  
  An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations Silvia Paracchini¹, Anthony P. Monaco¹, Julian C. Knight^{1 1}Wellcome Trust Centre for Human Genetics, University of Oxford Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

• Neuroscience

  
  Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound (fTCD) Dorothy V. M. Bishop¹, Nicholas A. Badcock¹, Georgina Holt^{1 1}Department of Experimental Psychology, University of Oxford Functional transcranial Doppler sonography (fTCD) is a simple and non-invasive ultrasound technique which can be used to assess the lateralization of cognitive functions, especially language, and is suitable for use with children.

• Biology

  
  Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy Ian M. Dobbie¹, Alexander Robson², Nicolas Delalez², Mark C. Leake^{2 1}Biochemistry, University of Oxford, ²Physics, University of Oxford Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

• Biology

  
  Focal Ca²⁺ Transient Detection in Smooth Muscle John S. Young¹, Robert J. Amos¹, Keith L. Brain^{1 1}Department of Pharmacology, University of Oxford Details methods for high-resolution Ca²⁺ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.