University of Oxford View Institution's Website 65 articles published in JoVE Biochemistry Analysis of Protein Complex Formation at Micromolar Concentrations by Coupling Microfluidics with Mass Photometry Myndert Claasen1, Zornitsa Kofinova1, Matteo Contino2, Weston B. Struwe3,4 1Refeyn Ltd., 2Adaptix Ltd., 3Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, 4The Kavli Institute for Nanoscience Discovery, University of Oxford This protocol combines mass photometry with a novel microfluidics system to investigate low-affinity protein-protein interactions. This approach is based on the rapid dilution of highly concentrated complexes in solution, which enables low-affinity measurements and broadens the applicability of mass photometry. Biology Dual-Dye Optical Mapping of Hearts from RyR2R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia Yangpeng Li*1,2, Jun Yang*1, Rui Zhang1, Tangting Chen1, Shiyu Zhang1, Yuqing Zheng1, Qiang Wen3, Tao Li1, Xiaoqiu Tan1,2, Ming Lei1,4, Xianhong Ou1 1Key Laboratory of Medical Electrophysiology of Ministry of Education, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease, Institute of Cardiovascular Research, Southwest Medical University, 2Department of Cardiology, the Affiliated Hospital of Southwest Medical University, 3Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 4Department of Pharmacology, University of Oxford This protocol introduces dual-dye optical mapping of mouse hearts obtained from wild-type and knock-in animals affected by catecholaminergic polymorphic ventricular tachycardia, including electrophysiological measurements of transmembrane voltage and intracellular Ca2+ transients with high temporal and spatial resolution. Biochemistry High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies Sagar Raturi1, Huanyu Li1, Yung-Ning Chang2, Andreea Scacioc1, Tina Bohstedt1, Alejandra Fernandez-Cid1, Adam Evans1, Patrizia Abrusci1, Abilasha Balakrishnan1, Tomas C. Pascoa1, Didi He1, Gamma Chi1, Nanki Kaur Singh1, Mingda Ye1, Anna Li1, Leela Shrestha1, Dong Wang1, Eleanor P. Williams1, Nicola A. Burgess-Brown1, Katharina L. Dürr1, Vera Puetter2, Alvaro Ingles-Prieto3, David B. Sauer1 1Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, 2Nuvisan ICB GmbH, 3CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences Structural and biochemical studies of human membrane transporters require milligram quantities of stable, intact, and homogeneous protein. Here we describe scalable methods to screen, express, and purify human solute carrier transporters using codon-optimized genes. Neuroscience Histological Examination of Mitochondrial Morphology in a Parkinson's Disease Model Dalila Ciceri*1, Lierni Gregorio-Zabala*1, Xabier Llama-Pino1, Begüm Kurt1, Jon Olano-Bringas1, Patricia Villegas-Zafra1, Nora Bengoa-Vergniory1,2,3,4 1Achucarro Basque Center for Neuroscience, 2Department of Neuroscience, University of the Basque Country (UPV/EHU), 3Ikerbasque - Basque Foundation for Science, 4Oxford Parkinson’s Disease Centre and Department of Physiology, Anatomy and Genetics, University of Oxford This study presents a method to analyze the morphology of mitochondria based on immunostaining and image analysis in mouse brain tissue in situ. It also describes how this allows one to detect changes in mitochondrial morphology induced by protein aggregation in Parkinson's disease models. Biology Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures Hwei Minn Khoo*1, Grace A. Meaker*1, Adam C. Wilkinson1 1MRC Weatherall Institute of Molecular Medicine, University of Oxford Presented here is a protocol to initiate, maintain, and analyze mouse hematopoietic stem cell cultures using ex vivo polyvinyl alcohol-based expansion, as well as methods to genetically manipulate them by lentiviral transduction and electroporation. Cancer Research Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma Kel Vin Tan*1,2, Xinxiang Yang*3, Chung Ying Chan2, Jingjing Shi1, Hing-Chiu Chang4, Keith Wan-Hang Chiu1,5, Kwan Man3 1Department of Diagnostic Radiology, School of Clinical Medicine, LKS Faculty of Medicine, The University of Hong Kong, 2Department of Oncology, MRC Oxford Institute for Radiation Oncology, University of Oxford, 3Department of Surgery, School of Clinical Medicine, HKU-SZH & LKS Faculty of Medicine, The University of Hong Kong, 4Department of Biomedical Engineering, The Chinese University of Hong Kong, 5Department of Diagnostic and Interventional Radiology, Kwong Wah Hospital Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG). Biology Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging Yuewen Sheng1, Kyle Morris1, Julika Radecke*1, Peijun Zhang*1,2,3 1Electron 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 Ayaz1,2, Jade Bailey1,2, Andrew Shaw1,2, Eileen McNeill1,2, Ben Davies1,2, Keith M. Channon1,2, Mark J. Crabtree1,2 1BHF 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. Dustin1 1Kennedy 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 Adams2,3, Stephen Lofts2, Minjeong Kwak4, Tae Geol Lee4, Colin Johnston5, Stephan Wagner6, Timothy R. Holbrook6, Yves U. Hachenberger7, Jutta Tentschert7, Nicholas Davidson1, Eugenia Valsami-Jones1 1School 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. Chemistry Achieving Efficient Fragment Screening at XChem Facility at Diamond Light Source Alice Douangamath*1,2, Ailsa Powell*1,2, Daren Fearon*1,2, Patrick M. Collins1,2, Romain Talon1,2,3, Tobias Krojer3,4, Rachael Skyner1,2, Jose Brandao-Neto1,2, Louise Dunnett1,2, Alexandre Dias1,2, Anthony Aimon1,2,3, Nicholas M. Pearce1,3, Conor Wild3,5, Tyler Gorrie-Stone1, Frank von Delft1,2,3,4,6 1Diamond Light Source Ltd, Harwell Science and Innovation Campus, 2Research Complex at Harwell, Harwell Science and Innovation Campus, 3Structural Genomics Consortium, University of Oxford, 4Centre for Medicines Discovery, University of Oxford, 5Oxford Protein Informatics Group, Department of Statistics, Oxford University, 6Department of Biochemistry, University of Johannesburg This paper describes the complete XChem process for crystal-based fragment screening, starting from applying for access and all subsequent steps to data dissemination. 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. Nahas1,2, Archana Jadhav1, Mohamed A. Koronfel1, Johannes Groen3, Eva Pereiro3, Ian M. Dobbie4, Maria Harkiolaki1 1Harwell 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 Stride1 1Institute 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. Biochemistry Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time Samuel G. Usher1, Frances M. Ashcroft1, Michael C. Puljung1,2 1Department of Physiology, Anatomy and Genetics, University of Oxford, 2Department of Chemistry and Neuroscience Program, Trinity College This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as Förster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid. Neuroscience In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages Hazel Hall-Roberts1,2,3, Elena Di Daniel2, William S. James1, John B. Davis2, Sally A. Cowley1 1James 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 Yoon6,7, Colin Johnston3, Stephen Lofts8, Eugenia Valsami-Jones1 1School 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 Matsuda1,2, Takako Koujin1, Lothar Schermelleh3, Tokuko Haraguchi1,2, Yasushi Hiraoka1,2 1Advanced 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 Monteiro1,2,3 1MRC 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 Hamilton1,2, Elisa Vergari1, Caroline Miranda3, Andrei I. Tarasov1,4,5 1Oxford 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 Desebrock1 1Department 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 Jarvis1 1Department of Plant Sciences, University of Oxford 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. 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 Gérard1 1The 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 Tan1,2,4, Wesley Thevathasan2,3,4,5, Jennifer McGinley6, Peter Brown7, Thushara Perera1,4 1Department 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 Fernandes1,2, Anneke Janzen1, Ayushi Nayak1, Jillian Swift1, Jana Zech1, Nicole Boivin1, Patrick Roberts1 1Max 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. Bott1,3, Kenneth H. Fischbeck1, Carlo Rinaldi1,4 1Neurogenetics 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-Parra1,2,3,4 1Cellular 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. Lee1 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 Escudero1,2, R Craig MacLean3, Alvaro San Millan4 1Department 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. Ishmukhametov2 1First 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. Wolfes1,2,3, Camin Dean3 1Chemical 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. Vincent1 1Department 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. Fang1,6, Konstantinos Palikaras2, Nuo Sun3, Elayne M. Fivenson1, Ryan D. Spangler4, Jesse S. Kerr1, Stephanie A. Cordonnier1, Yujun Hou1, Eszter Dombi5, Henok Kassahun6, Nektarios Tavernarakis2,7, Joanna Poulton5, Hilde Nilsen6, Vilhelm A. Bohr1,8 1Laboratory 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 Shpektor1,3, Maria Nazarova4, Matteo Feurra1,2 1School 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 Abbey2 1ARC 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 Moore1 1Department 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 Bushell4 1Division 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 Jarvis1 1Department 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 Sunter1 1Sir 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. FitzGerald2 1Nuffield 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. Baczynska1,2, Sarah Shaw3, Helen C. Roberts1,2,3,5, Cyrus Cooper2,3,4, Avan Aihie Sayer1,2,3,5,6, Harnish P. Patel1,2,3 1Academic 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 Kartje4,5,6, John S. Beech7, Diana Cash2, Lawrence Moon1 1Department 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 Aigrain1,2, Marko Sustarsic1, Robert Crawford1, Anne Plochowietz1, Achillefs N. Kapanidis1 1Clarendon 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. Preston1 1Department 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. Bowden1 1Oxford 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 Monico1,2, Gionata Belcastro1, Francesco Vanzi1,3, Francesco S. Pavone1,4,5,6, Marco Capitanio1,4 1LENS - 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 Brown1 1Nuffield 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. Watkins1 1Department 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. Gilbert1 1Division 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 Uphoff1,2, David J. Sherratt1, Achillefs N. Kapanidis2 1Microbiology 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. Carvalho1,2, Derric Nimmo1, Neil Naish1, Andrew R. McKemey1, Pam Gray1, André B. B. Wilke3, Mauro T. Marrelli3, Jair F. Virginio4, Luke Alphey1,5, Margareth L. Capurro2,6 1Oxitec 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. Cox1 1Department 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. Deacon1 1Department 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. Deacon1 1Department 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. Deacon1 1Department 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. Deacon1 1Department 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 Davis1 1Department 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 Deacon1 1Department 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. Slater1,4 1Neuroscience, 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 Niedzwiedz1,2 1Department 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. Deacon1 1Department 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. Knight1 1Wellcome 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 Holt1 1Department 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. Leake2 1Biochemistry, 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. Brain1 1Department 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.
