University of Bristol View Institution's Website 25 articles published in JoVE Biology Zebrafish Scale Regeneration In Toto and Ex Vivo Culture of Scales Qiao Tong*1, Renata Raele*1, Dylan Bergen1, Chrissy Hammond1 1School of Physiology, Pharmacology and Neuroscience, Biomedical Science Building, University of Bristol This protocol describes the harvesting and visualization of elasmoid scales of zebrafish during in vivo regeneration. In addition, the ex vivo culture of these scales for up to 7 days after harvest is presented. Medicine Local Anesthetic Thoracoscopy for Undiagnosed Pleural Effusion Uffe Bodtger1,2, José M. Porcel3, Rahul Bhatnagar4,5, Nick Maskell4,5, Mohammed Munavvar6,7, Casper Jensen1, Paul Frost Clementsen1,8, Daniel Bech Rasmussen1,2 1Respiratory Research Unit PLUZ, Department of Respiratory Medicine, Zealand University Hospital, 2Institute of Regional Health Research, University of Southern Denmark, 3Pleural Medicine Unit, Department of Internal Medicine, Hospital Universitari Arnau de Vilanova, IRBLleida, 4Respiratory Department, Southmead Hospital, North Bristol NHS Trust, 5Academic Respiratory Unit, University of Bristol, 6Lancashire Teaching Hospitals, 7University of Central Lancashire, 8Centre for HR and Education, Copenhagen Academy for Medical Education and Simulation Local anesthetic thoracoscopy (LAT) is essential for diagnosing recurrent, undiagnosed pleural effusion when a guideline-based workup fails to provide a specific cause. LAT can be performed as a day-case procedure by chest physicians. Here, we present a step-by-step approach for a successful and safe procedure. Neuroscience Open-Source Real-Time Closed-Loop Electrical Threshold Tracking for Translational Pain Research Aidan P. Nickerson1,2, Graeme W. T. Newton1, James H. O'Sullivan3, Manuel Martinez-Perez4, Anna C. Sales1, Gethin Williams5, Anthony E. Pickering1, James P. Dunham1 1Anaesthesia, Pain, and Critical Care Sciences, School of Physiology, Pharmacology, & Neuroscience, University of Bristol, 2Eli Lilly and Company, 3Department of Computer Science, University of Bristol, 4Department of Aerospace Engineering, University of Bristol, 5Research Computing, University of Bristol APTrack is a software plugin developed for the Open Ephys platform that enables real-time data visualization and the closed-loop electrical threshold tracking of neuronal action potentials. We have successfully used this in microneurography for human C-fiber nociceptors and mouse C-fiber and Aδ-fiber nociceptors. Engineering Microfluidic Device for the Separation of Non-Metastatic (MCF-7) and Non-Tumor (MCF-10A) Breast Cancer Cells Using AC Dielectrophoresis Ateeq ur Rehman1, Rahman S. Zabibah2, Samira Kharratian3, Adil Mustafa4,5 1Department of Biomedical Engineering, Foundation University Islamabad, 2College of Medical Technology, The Islamic University Najaf, 3Faculty of Mechanical Engineering, University of Tabriz, 4Department of Engineering Mathematics, University of Bristol, 5School of Life Sciences, University of Warwick Breast cancer cells exhibit different dielectric properties compared to non-tumor breast epithelial cells. It has been hypothesized that, based on this difference in dielectric properties, the two populations can be separated for immunotherapy purposes. To support this, we model a microfluidic device to sort MCF-7 and MCF-10A cells. Neuroscience Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex Lisa Kinnavane1, Paul J. Banks1 1School of Physiology, Pharmacology and Neuroscience, University of Bristol Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices. Immunology and Infection Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis Sarah John1, Oliver H. Bell2, Leslie Wilson1, David A. Copland2, Kathryn L. Pepple1 1Department of Ophthalmology, University of Washington, 2Academic Unit of Ophthalmology, Translational Health Sciences, University of Bristol This protocol outlines the steps for inducing Primed Mycobacterial Uveitis (PMU) in mice. This method outlines the steps to help produce reliable and robust ocular inflammation in the mouse model system. Using this protocol, we generated uveitic eyes and uninflamed fellow eyes from single animals for further evaluation with immunologic, transcriptomic, and proteomic assays. Biochemistry PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays James W. Saville1, Luca A. Troman2, Franck Duong Van Hoa1 1Department of Biochemistry, University of British Columbia, 2School of Biochemistry, University of Bristol We present a method that combines membrane protein purification and reconstitution into peptidiscs in a single chromatographic step. Biotinylated scaffolds are used for direct surface attachment and measurement of protein-ligand interactions via biolayer interferometry. Bioengineering CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art Klaudia Ciurkot1,2, Brenda Vonk1, Thomas E. Gorochowski3,4, Johannes A. Roubos1, René Verwaal1 1DSM Biotechnology Center, 2Biochemistry and Molecular Biology, Department of Chemistry, University of Hamburg, 3BrisSynBio, University of Bristol, Life Sciences Building, 4School of Biological Sciences, University of Bristol, Life Sciences Building The CRISPR/Cas12a system in combination with a single crRNA array enables efficient multiplex editing of the S. cerevisiae genome at multiple loci simultaneously. This is demonstrated by constructing carotenoid producing yeast strains which are subsequently used to create yeast pixel art. Medicine A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence Eric Parent1, Patrick Burns2, André Desrochers1, Martine Boulianne1 1Department of Clinical Sciences, Faculty of Veterinary Medicine, Université de Montréal, 2University of Bristol Here we present a protocol to surgically create 'intestinal ligated loops' in chicken small intestines. This procedure allows for the comparison of multiple Clostridium perfringens strains' virulence in situ in a single host. This method markedly decreases the number of chickens usually necessary for similar in vivo experiments. Medicine Assessment of Kidney Function in Mouse Models of Glomerular Disease Megan Stevens1,2,3, Sebastian Oltean1,2,3 1Institute of Biomedical and Clinical Sciences, Medical School, University of Exeter, 2School of Physiology, Pharmacology and Neurosciences, University of Bristol, 3Bristol Renal, School of Clinical Sciences, University of Bristol This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease. Immunology and Infection Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae Helen Weavers1,2, Anna Franz1, Will Wood3, Paul Martin1,4 1School of Biochemistry, Biomedical Sciences, University of Bristol, 2School of Cellular and Molecular Medicine, Biomedical Sciences, University of Bristol, 3MRC Centre for Inflammation Research, University of Edinburgh, Queens Medical Research Institute, 4School of Physiology, Pharmacology, and Neuroscience, Biomedical Sciences, University of Bristol Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation. Cancer Research Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy Vaibhav Murthy*1, Dalton Dacus*1, Monica Gamez2, Changkun Hu1, Sebastian O. Wendel1, Jazmine Snow1, Andrew Kahn1, Stephen H. Walterhouse1, Nicholas A. Wallace1 1Division of Biology, Kansas State University, 2Bristol Medical School, Translational Health Sciences, University of Bristol Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism. Neuroscience A Magnetic Resonance Imaging Protocol for Stroke Onset Time Estimation in Permanent Cerebral Ischemia Bryony L. McGarry*1, Kimmo T. Jokivarsi*2, Michael J. Knight1, Olli H.J. Grohn2, Risto A. Kauppinen1 1School of Experimental Psychology and Clinical Research and Imaging Center Bristol, University of Bristol, 2Department of Neurobiology, A.I. Virtanen Institute, University of Eastern Finland A protocol for stroke onset time estimation in a rat model of stroke exploiting quantitative magnetic resonance imaging (qMRI) parameters is described. The procedure exploits diffusion MRI for delineation of the acute stroke lesion and quantitative T1 and T2 (qT1 and qT2) relaxation times for timing of stroke. Engineering Ice Generation and the Heat and Mass Transfer Phenomena of Introducing Water to a Cold Bath of Brine Xiao Yun1, Giuseppe L. Quarini1 1Department of Mechanical Engineering, University of Bristol Here, we present a protocol to demonstrate the generation of ice when water is introduced to a cold bath of brine, as a secondary refrigerant, at a range of temperatures well below the freezing point of water. It can be used as an alternative way of producing ice for industry. Biology A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix Andrew L. Hellewell1, Silvia Rosini1, Josephine C. Adams1 1School of Biochemistry, University of Bristol The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies. Bioengineering Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells Sara Correia Carreira1, James P.K. Armstrong2, Mitsuhiro Okuda3,4, Annela M. Seddon1, Adam W. Perriman5, Walther Schwarzacher6 1Bristol Centre for Functional Nanomaterials, University of Bristol, 2Department of Materials, Imperial College London, 3Self Assembly Group, CIC nanoGUNE, 4Ikebasque, Basque Foundation for Science, 5School of Cellular and Molecular Medicine, University of Bristol, 6H.H. Wills Physics Laboratory, University of Bristol A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin. Developmental Biology Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics Lucy H. Brunt1, Karen A. Roddy1, Emily J. Rayfield2, Chrissy L. Hammond1 1Physiology, Pharmacology and Neuroscience, University of Bristol, 2Earth Sciences, University of Bristol Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw. Developmental Biology Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients Simon W. Fellgett1, Simon A. Ramsbottom2, Richard J. Maguire3, Stephen Cross4, Peter O'Toole5, Mary E. Pownall5 1Department of Biomedical Science, The Bateson Centre, University of Sheffield, 2Institute of Genetic Medicine, Newcastle University, 3Department of Cardiovascular Science, The Bateson Centre, University of Sheffield, 4School of Biochemistry, University of Bristol, 5Biology Department, University of York The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients. Biology Comparing the Affinity of GTPase-binding Proteins using Competition Assays Rosalind C. Williamson1, Mark D. Bass2 1School of Biochemistry, University of Bristol, 2Department of Biomedical Science, University of Sheffield This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate. Neuroscience Drosophila Adult Olfactory Shock Learning Bilal R. Malik1, James J.L. Hodge1 1Physiology and Pharmacology, University of Bristol The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured. Biology Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening Marisa Till1, Alice Robson1, Matthew J. Byrne1, Asha V. Nair1, Stefan A. Kolek2, Patrick D. Shaw Stewart2, Paul R. Race1 1School of Biochemistry, University of Bristol, 2Douglas Instruments Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments. Bioengineering Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound James Roper1, Andrew Harrison2, Mark D. Bass1 1School of Biochemistry, University of Bristol, 2Smith and Nephew This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy. Neuroscience Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching Keri L. Hildick1, Inmaculada M. González-González1, Frédéric Jaskolski1, Jeremy. M. Henley1 1MRC Centre for Synaptic Plasticity, University of Bristol This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons. Immunology and Infection Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human α-actinin by Confocal Imaging Isabel Murillo1, Mumtaz Virji1 1Department of Cellular and Molecular Medicine, University of Bristol, UK Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human α-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular α-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs). Biology In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells Anyaporn Sawasdichai1, Hsin-Tien Chen1, Nazefah Abdul Hamid1, Padma-Sheela Jayaraman2, Kevin Gaston1 1Department of Biochemistry, School of Medical Sciences, University of Bristol, 2Division of Immunity and Infection, School of Medicine, University of Birmingham In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.
