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65 ARTICLES PUBLISHED IN JoVE

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Biology

Focal Ca2+ Transient Detection in Smooth Muscle
John S. Young 1, Robert J. Amos 1, Keith L. Brain 1
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.

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Biology

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy
Ian M. Dobbie 1, Alexander Robson 2, Nicolas Delalez 2, Mark C. Leake 2
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.

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Neuroscience

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound (fTCD)
Dorothy V. M. Bishop 1, Nicholas A. Badcock 1, Georgina Holt 1
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.

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Biology

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations
Silvia Paracchini 1, Anthony P. Monaco 1, Julian C. Knight 1
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.

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Behavior

Shallow Water (Paddling) Variants of Water Maze Tests in Mice
Robert M.J. Deacon 1
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.

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Neuroscience

Measuring Motor Coordination in Mice
Robert M.J. Deacon 1
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.

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Medicine

Measuring the Strength of Mice
Robert M.J. Deacon 1
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.

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Neuroscience

Hyponeophagia: A Measure of Anxiety in the Mouse
Rob M.J. Deacon 1
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

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Behavior

The Successive Alleys Test of Anxiety in Mice and Rats
Robert M.J. Deacon 1
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.

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Immunology and Infection

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction
Xin Meng 1, Gongpu Zhao 1, Peijun Zhang 1
1Department of Structural Biology, University of Pittsburgh School of Medicine

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

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Neuroscience

Electrophysiological Measurements and Analysis of Nociception in Human Infants
L. Fabrizi *1, A. Worley *2, D. Patten 1, S. Holdridge 1, L. Cornelissen 1, J. Meek 3, S. Boyd 2, R. Slater 1,4
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.

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Environment

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil
Danilo O. Carvalho 1,2, Derric Nimmo 1, Neil Naish 1, Andrew R. McKemey 1, Pam Gray 1, André B. B. Wilke 3, Mauro T. Marrelli 3, Jair F. Virginio 4, Luke Alphey 1,5, Margareth L. Capurro 2,6
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.

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Bioengineering

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions
Sangmi Jun 1, Gongpu Zhao 1, Jiying Ning 1, Gregory A. Gibson 2, Simon C. Watkins 2, Peijun Zhang 1
1Department of Structural Biology, University of Pittsburgh School of Medicine, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

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Biology

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases
Penelope A. Mason 1, Ivan Boubriak 1, Lynne S. Cox 1
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.

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Immunology and Infection

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking
Stephan Uphoff 1,2, David J. Sherratt 1, Achillefs N. Kapanidis 2
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.

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Biology

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies
Luke A. Yates 1, Robert J. C. Gilbert 1
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.

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Medicine

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation
Simon Little 1, Alek Pogosyan 1, Spencer Neal 2, Ludvic Zrinzo 2, Marwan Hariz 2, Thomas Foltynie 2, Patricia Limousin 2, Peter Brown 1
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.

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Biology

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions
Carina Monico 1,2, Gionata Belcastro 1, Francesco Vanzi 1,3, Francesco S. Pavone 1,4,5,6, Marco Capitanio 1,4
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.

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Behavior

Stimulating the Lip Motor Cortex with Transcranial Magnetic Stimulation
Riikka Möttönen 1, Jack Rogers 1, Kate E. Watkins 1
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.

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Immunology and Infection

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo
Juha T. Huiskonen 1, Marie-Laure Parsy 1, Sai Li 1, David Bitto 1, Max Renner 1, Thomas A. Bowden 1
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.

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Biology

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example
Brendan M. O'Leary 1, Arantza Rico 2, Sarah McCraw 1, Helen N. Fones 3, Gail M. Preston 1
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.

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Biology

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation
Louise Aigrain 1,2, Marko Sustarsic 1, Robert Crawford 1, Anne Plochowietz 1, Achillefs N. Kapanidis 1
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.

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Medicine

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome
Alicja M. Baczynska 1,2, Sarah Shaw 3, 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
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.

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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. Roy 3, Barbara Haenzi 1, Shi-Yen Tsai 4, Gwendolyn Kartje 4,5,6, John S. Beech 7, Diana Cash 2, Lawrence Moon 1
1Wolfson Centre for Age-Related Diseases, King's College London, University of London, 2Department 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.

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Immunology and Infection

High-throughput Gene Tagging in Trypanosoma brucei
Philip Dyer 1, Samuel Dean 1, Jack Sunter 1
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.

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JoVE Journal

Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants
Qihua Ling 1, Paul Jarvis 1
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.

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Immunology and Infection

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry
Stephen C. Juvet 1, Sajad Moshkelgosha 2, Sharon Sanderson 3, Joanna Hester 4, Kathryn J. Wood 4, Andrew Bushell 4
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.

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Developmental Biology

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development
Charlotte Kirchhelle 1, Ian Moore 1
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.

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Genetics

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents
Naemeh Pourshafie 1, Philip R. Lee 2, Ke-lian Chen 1, George G. Harmison 1, Laura C. Bott 1,3, Kenneth H. Fischbeck 1, Carlo Rinaldi 1,4
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.

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JoVE Core

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation
Anna Shpektor 1,3, Maria Nazarova 4, Matteo Feurra 1,2
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.

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Neuroscience

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol
Anne C. Wolfes 1,2,3, Camin Dean 3
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.

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Chemistry

Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene
Rebecca A. Ryan 1, Sophie Williams 1, Andrew V. Martin 1, Ruben A. Dilanian 1, Connie Darmanin 2, Corey T. Putkunz 1, David Wood 3, Victor A. Streltsov 4, Michael W.M. Jones 5, Naylyn Gaffney 6, Felix Hofmann 7, Garth J. Williams 8, Sebastien Boutet 9, Marc Messerschmidt 10, M. Marvin Seibert 11, Evan K. Curwood 11, Eugeniu Balaur 2, Andrew G. Peele 5, Keith A. Nugent 2, Harry M. Quiney 1, Brian Abbey 2
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.

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Medicine

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice
Evandro F. Fang 1,6, Konstantinos Palikaras 2, Nuo Sun 3, Elayne M. Fivenson 1, Ryan D. Spangler 4, Jesse S. Kerr 1, Stephanie A. Cordonnier 1, Yujun Hou 1, Eszter Dombi 5, Henok Kassahun 6, Nektarios Tavernarakis 2,7, Joanna Poulton 5, Hilde Nilsen 6, Vilhelm A. Bohr 1,8
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.

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JoVE Journal

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance
Jose Antonio Escudero 1,2, R Craig MacLean 3, Alvaro San Millan 4
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.

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Biochemistry

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis
Alicia Ventresca Miller 1, Ricardo Fernandes 1,2, Anneke Janzen 1, Ayushi Nayak 1, Jillian Swift 1, Jana Zech 1, Nicole Boivin 1, Patrick Roberts 1
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.

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Biochemistry

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software
Rory Nolan 1, Luis A. Alvarez 1, Samuel C. Griffiths 2, Jonathan Elegheert 2, Christian Siebold 2, Sergi Padilla-Parra 1,2,3,4
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.

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Immunology and Infection

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins
Meisam Naeimi Kararoudi 1, Hamid Dolatshad 2, Prashant Trikha 1, Syed-Rehan A. Hussain 3, Ezgi Elmas 1, Jennifer A. Foltz 1, Jena E. Moseman 1, Aarohi Thakkar 1, Robin J. Nakkula 1, Margaret Lamb 1, Nitin Chakravarti 1, K. John McLaughlin 3, Dean A. Lee 1
1Center for Childhood Cancer and Blood Disease, Nationwide Children's Hospital, 2Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, 3Center for Clinical and Translational Research, Nationwide Children's Hospital Research Institute

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).

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JoVE Core

An Instrumented Pull Test to Characterize Postural Responses
Joy Tan 1,2,4, Wesley Thevathasan 2,3,4,5, Jennifer McGinley 6, Peter Brown 7, Thushara Perera 1,4
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.

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Biology

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells
Alexander Hamilton 1,2, Elisa Vergari 1, Caroline Miranda 3, Andrei I. Tarasov 1,4,5
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.

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Immunology and Infection

Tuning Degradation to Achieve Specific and Efficient Protein Depletion
J. David Barrass 1, Gonzalo I. Mendoza-Ochoa 1,2, Isabella E. Maudlin 1,3, Emanuela Sani 1, Jean D. Beggs 1
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.

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JoVE Journal

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

Chloroplast Research Methods: Probing The Targeting, Localization And Interactions Of Chloroplast Proteins

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Behavior

Investigating the Effect of Visual Imagery and Learning Shape-Audio Regularities on Bouba and Kiki
Torø Graven 1, Clea Desebrock 1
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.

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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
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.

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Biology

High-Accuracy Correction of 3D Chromatic Shifts in the Age of Super-Resolution Biological Imaging Using Chromagnon
Atsushi Matsuda 1,2, Takako Koujin 1, Lothar Schermelleh 3, Tokuko Haraguchi 1,2, Yasushi Hiraoka 1,2
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.

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Chemistry

Easy Access to Aliphatic Sulfonamides using Sulfamoyl Chlorides Under Visible Light Activation
Sandrine M. Hell 1, Claudio F. Meyer 1,2, Andrés A. Trabanco 2, Véronique Gouverneur 1
1Chemistry Research Laboratory, University of Oxford, 2Discovery Chemistry, Janssen Research and Development

Presented here is a protocol for the easy synthesis of aliphatic sulfonamides using sulfamoyl chlorides, (TMS)3SiH and Eosin Y under blue-light irradiation.

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Biochemistry

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time
Samuel G. Usher 1, Frances M. Ashcroft 1, Michael C. Puljung 1,2
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.

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Biology

Preparation of a User-Defined Peptide Gel for Controlled 3D Culture Models of Cancer and Disease
Jennifer C. Ashworth 1, Rebecca L. Morgan 1, Kataryna Lis-Slimak 1, Kate A. Meade 1, Sal Jones 1, Katherine Spence 2, Charlotte E. Slater 1, Jamie L. Thompson 1, Anna M. Grabowska 1, Robert B. Clarke 2, Gilian Farnie 3, Cathy L. R. Merry 1
1Division of Cancer & Stem Cells, School of Medicine, Nottingham Biodiscovery Institute, University of Nottingham, 2Breast Biology Group, Manchester Breast Centre, Division of Cancer Sciences, Faculty of Biology, Medicine and Health, University of Manchester, 3SGC, Botnar Research Centre, NDORMS, University of Oxford

We present a method for creating a 3D cell culture environment, which can be used to investigate the importance of cell/matrix interactions in cancer progression. Using a simple self-assembling octapeptide, the matrix surrounding encapsulated cells can be controlled, with independent regulation of mechanical and biochemical cues.

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Chemistry

Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison
Sophie M. Briffa 1, Jo Sullivan 2, Agnieszka Siupa 2, Pauline Carnell-Morris 2, Michele Carboni 2, Kerstin Jurkschat 3, Ruud J. B. Peters 4, Carolin Schultz 5, Kang Hee Seol 6, Sook-Jin Kwon 6, Sehee Park 7, Tae Hyun Yoon 6,7, Colin Johnston 3, Stephen Lofts 8, Eugenia Valsami-Jones 1
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.

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Engineering

UV-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media
Ana C. Quevedo 1, Emily Guggenheim 1, Sophie M. Briffa 1, Jessica Adams 2,3, Stephen Lofts 2, Minjeong Kwak 4, Tae Geol Lee 4, Colin Johnston 5, Stephan Wagner 6, Timothy R. Holbrook 6, Yves U. Hachenberger 7, Jutta Tentschert 7, Nicholas Davidson 1, Eugenia Valsami-Jones 1
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.

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Biology

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation
Naveed Akbar 1, Katherine E. Pinnick 2, Daan Paget 1, Robin P. Choudhury 1
1Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, 2Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford

We describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

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Bioengineering

Studying Cavitation Enhanced Therapy
Michael Gray *1, Alexandra V. Vasilyeva *1, Veerle Brans *1, Eleanor Stride 1
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.

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Neuroscience

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages
Hazel Hall-Roberts 1,2,3, Elena Di Daniel 2, William S. James 1, John B. Davis 2, Sally A. Cowley 1
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.

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Biology

Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells
Nina Vyas *1, Nina Perry *1, Chidinma A. Okolo 1, Ilias Kounatidis 1, Thomas M. Fish 1, Kamal L. Nahas 1,2, Archana Jadhav 1, Mohamed A. Koronfel 1, Johannes Groen 3, Eva Pereiro 3, Ian M. Dobbie 4, Maria Harkiolaki 1
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.

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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. Collins 1,2, Romain Talon 1,2,3, Tobias Krojer 3,4, Rachael Skyner 1,2, Jose Brandao-Neto 1,2, Louise Dunnett 1,2, Alexandre Dias 1,2, Anthony Aimon 1,2,3, Nicholas M. Pearce 1,3, Conor Wild 3,5, Tyler Gorrie-Stone 1, Frank von Delft 1,2,3,4,6
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.

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Biology

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging
Qing Liu *1,2, Kel Vin Tan *3, Hing-Chiu Chang 3, Pek-Lan Khong 3, Xiaoyan Hui 1,2,4
1State Key Laboratory of Pharmaceutical Biotechnology, The University of Hong Kong, 2Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 3Department of Diagnostic Radiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 4School of Biomedical Sciences, Institute of Vascular Medicine, Chinese University of Hong Kong

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

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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
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.

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Biology

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging
Yuewen Sheng 1, Kyle Morris 1, 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.

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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 Chan 2, Jingjing Shi 1, Hing-Chiu Chang 4, Keith Wan-Hang Chiu 1,5, Kwan Man 3
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).

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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. Wilkinson 1
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.

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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 Zhang 1, Tangting Chen 1, Shiyu Zhang 1, Yuqing Zheng 1, Qiang Wen 3, Tao Li 1, Xiaoqiu Tan 1,2, Ming Lei 1,4, Xianhong Ou 1
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.

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Neuroscience

Histological Examination of Mitochondrial Morphology in a Parkinson's Disease Model
Dalila Ciceri *1, Lierni Gregorio-Zabala *1, Xabier Llama-Pino 1, Begüm Kurt 1, Jon Olano-Bringas 1, Patricia Villegas-Zafra 1, Nora Bengoa-Vergniory 1,2,3,4
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.

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Genetics

CATCH-UP: A High-Throughput Upstream-Pipeline for Bulk ATAC-Seq and ChIP-Seq Data
Simone G. Riva 1,2, Emily Georgiades 1,2, E. Ravza Gur 1,2, Matthew Baxter 1,2,3, Jim R. Hughes 1,2
1MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, 2MRC WIMM Centre for Computational Biology, MRC Weatherall Institute of Molecular Medicine, University of Oxford, 3Division of Cardiovascular Medicine, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford

ATAC-seq and ChIP-seq allow detailed investigation of gene regulation; however, processing these data types is challenging and often inconsistent between research groups. We present CATCH-UP: an easy-to-use computational pipeline that allows standardized and reproducible data processing and analysis of new and published ATAC/ChIP-seq datasets.

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Biochemistry

Analysis of Protein Complex Formation at Micromolar Concentrations by Coupling Microfluidics with Mass Photometry
Myndert Claasen 1, Zornitsa Kofinova 1, Matteo Contino 2, Weston B. Struwe 3,4
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.

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Biochemistry

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies
Sagar Raturi 1, Huanyu Li 1, Yung-Ning Chang 2, Andreea Scacioc 1, Tina Bohstedt 1, Alejandra Fernandez-Cid 1, Adam Evans 1, Patrizia Abrusci 1, Abilasha Balakrishnan 1, Tomas C. Pascoa 1, Didi He 1, Gamma Chi 1, Nanki Kaur Singh 1, Mingda Ye 1, Anna Li 1, Leela Shrestha 1, Dong Wang 1, Eleanor P. Williams 1, Nicola A. Burgess-Brown 1, Katharina L. Dürr 1, Vera Puetter 2, Alvaro Ingles-Prieto 3, David B. Sauer 1
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.

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Biochemistry

Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG
Daniel Ramírez Montero 1, Humberto Sánchez 1, Edo van Veen 1, Theo van Laar 1, Belén Solano 1, John F. X. Diffley 2, Nynke H. Dekker 1,3
1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, 2Chromosome Replication Laboratory, Francis Crick Institute, 3Department of Physics and Kavli Institute of Nanoscience Discovery, University of Oxford

We report a hybrid ensemble and single-molecule assay to directly image and quantify the motion of fluorescently labeled, fully reconstituted Cdc45/Mcm2-7/GINS (CMG) helicase on linear DNA molecules held in place in an optical trap.

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