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University of Bristol

28 ARTICLES PUBLISHED IN JoVE

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Biology

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells
Anyaporn Sawasdichai 1, Hsin-Tien Chen 1, Nazefah Abdul Hamid 1, Padma-Sheela Jayaraman 2, Kevin Gaston 1
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.

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Neuroscience

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching
Keri L. Hildick 1, Inmaculada M. González-González 1, Frédéric Jaskolski 1, Jeremy. M. Henley 1
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.

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Bioengineering

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound
James Roper 1, Andrew Harrison 2, Mark D. Bass 1
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.

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Neuroscience

Drosophila Adult Olfactory Shock Learning
Bilal R. Malik 1, James J.L. Hodge 1
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.

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Biology

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening
Marisa Till 1, Alice Robson 1, Matthew J. Byrne 1, Asha V. Nair 1, Stefan A. Kolek 2, Patrick D. Shaw Stewart 2, Paul R. Race 1
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.

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Developmental Biology

Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings
Patrick Burns 1, Hai Lun Liu 2, Shikha Kuthiala 2, Gilles Fecteau 1, André Desrochers 1, Lucien Daniel Durosier 2, Mingju Cao 2, Martin G. Frasch 2,3,4
1Département de sciences cliniques, CHUV, Université de Montréal, St-Hyacinthe, QC, 2Département d'obstetriques et de gynécologie, CHU Ste-Justine Research Centre, Université de Montréal, 3Département de neurosciences, CHU Ste-Justine Centre de recherche, Université de Montréal, 4Centre de recherche en reproduction animale (CRRA), Université de Montréal, St-Hyacinthe, QC

The chronically instrumented non-anesthetized fetal sheep model is used to study human fetal development in health and disease, because it permits surgical placement and maintenance of catheters and electrodes, repetitive blood sampling, substance injection, recording of bioelectrical activity, and in vivo imaging. We describe the procedures required to establish this model.

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Intra-articular injection of murine MSC for the treatment of inflammation and tissue degradation in Antigen-induced Arthritis.
Alasdair Kay 1, Jim Middleton 2, Oksana Kehoe 3
1Institute for Science and Technology in Medicine, Keele University, 2Faculty of Medicine and Dentistry, University of Bristol, 3Keele University

Here, we present a protocol to induce inflammatory arthritis in vivo using antigen response, termed Antigen-induced Arthritis (AIA), an effective tool for research into Rheumatoid Arthritis (RA) research. Additionally we demonstrate the therapeutic effects of infusion of MSC to reduce inflammation, cartilage degradation and expression of TNFα.

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Developmental Biology

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients
Simon W. Fellgett 1, Simon A. Ramsbottom 2, Richard J. Maguire 3, Stephen Cross 4, Peter O'Toole 5, Mary E. Pownall 5
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.

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Biology

Comparing the Affinity of GTPase-binding Proteins using Competition Assays
Rosalind C. Williamson 1, Mark D. Bass 2
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.

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Bioengineering

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells
Sara Correia Carreira 1, James P.K. Armstrong 2, Mitsuhiro Okuda 3,4, Annela M. Seddon 1, Adam W. Perriman 5, Walther Schwarzacher 6
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.

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JoVE Journal

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics
Lucy H. Brunt 1, Karen A. Roddy 1, Emily J. Rayfield 2, Chrissy L. Hammond 1
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.

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JoVE Core

Ice Generation and the Heat and Mass Transfer Phenomena of Introducing Water to a Cold Bath of Brine
Xiao Yun 1, Giuseppe L. Quarini 1
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.

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Biology

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix
Andrew L. Hellewell 1, Silvia Rosini 1, Josephine C. Adams 1
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.

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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. Knight 1, Olli H.J. Grohn 2, Risto A. Kauppinen 1
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.

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Medicine

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence
Eric Parent 1, Patrick Burns 2, André Desrochers 1, Martine Boulianne 1
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.

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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 Gamez 2, Changkun Hu 1, Sebastian O. Wendel 1, Jazmine Snow 1, Andrew Kahn 1, Stephen H. Walterhouse 1, Nicholas A. Wallace 1
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.

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Techniques to Assess Kidney Function in Mouse Models of Glomerular Disease
Megan Stevens 1,2,3, Sebastian Oltean 1,2,3
1School of Physiology, Pharmacology and Neurosciences, University of Bristol, 2Bristol Renal, School of Clinical sciences, University of Bristol, 3Institute of Biomedical and Clinical Sciences, Medical School, University of Exeter

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.

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Medicine

Assessment of Kidney Function in Mouse Models of Glomerular Disease
Megan Stevens 1,2,3, Sebastian Oltean 1,2,3
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.

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Immunology and Infection

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae
Helen Weavers 1,2, Anna Franz 1, Will Wood 3, Paul Martin 1,4
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.

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Biochemistry

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays
James W. Saville 1, Luca A. Troman 2, Franck Duong Van Hoa 1
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.

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Immunology and Infection

Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis
Sarah John 1, Oliver H. Bell 2, Leslie Wilson 1, David A. Copland 2, Kathryn L. Pepple 1
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.

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Neuroscience

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex
Lisa Kinnavane 1, Paul J. Banks 1
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.

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JoVE Core

Microfluidic Device for the Separation of Non-Metastatic (MCF-7) and Non-Tumor (MCF-10A) Breast Cancer Cells Using AC Dielectrophoresis
Ateeq ur Rehman 1, Rahman S. Zabibah 2, Samira Kharratian 3, Adil Mustafa 4,5
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.

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JoVE Core

Open-Source Real-Time Closed-Loop Electrical Threshold Tracking for Translational Pain Research
Aidan P. Nickerson 1,2, Graeme W. T. Newton 1, James H. O'Sullivan 3, Manuel Martinez-Perez 4, Anna C. Sales 1, Gethin Williams 5, Anthony E. Pickering 1, James P. Dunham 1
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.

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Biology

Zebrafish Scale Regeneration In Toto and Ex Vivo Culture of Scales
Qiao Tong *1, Renata Raele *1, Dylan Bergen 1, Chrissy Hammond 1
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.

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JoVE Core

Local Anesthetic Thoracoscopy for Undiagnosed Pleural Effusion
Uffe Bodtger 1,2, José M. Porcel 3, Rahul Bhatnagar 4,5, Nick Maskell 4,5, Mohammed Munavvar 6,7, Casper Jensen 1, Paul Frost Clementsen 1,8, Daniel Bech Rasmussen 1,2
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.

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Medicine

A Stepwise Approach for Performing Ultrasound Guided Transthoracic Lung Biopsy
Christian B. Laursen 1,2, Rahul Bhatnagar 2,3,4, Amanda Dandanell Juul 1,2
1Department of Respiratory Medicine, Odense University Hospital, 2Odense Respiratory Research Unit (ODIN), Department of Clinical Research, University of Southern Denmark, 3Respiratory Medicine Department, North Bristol NHS Trust, Southmead Hospital, 4Academic Respiratory Unit, University of Bristol

Transthoracic ultrasound-guided lung biopsy represents a safe, cost-effective, and efficient approach for patients presenting with subpleural lung lesions suspected of malignancy. Employing a systematic, step-by-step process is crucial to achieve optimal patient selection, minimize complication risks, and maximize diagnostic accuracy.

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Medicine

Amplitude-Integrated EEG in Infants at Risk of Hypoxic-Ischemic Encephalopathy: A Feasibility Study in Road and Air Transport in Western Australia
Alexander Wilson 1,2,3, Varuna Chaudhary 1,2,3, Ela Chakkrapani 4,5, Mary O'Dea 6,7, Jonathan Davis 1,2,3,8,9,10
1Government of Western Australia, Child and Adolescent Health Service, 2Newborn Emergency Transport Service Western Australia (NETS WA), 3Perth Children's Hospital, 4University of Bristol, 5St Michael's Hospital, 6Coombe Hospital, 7Children's Health Ireland, 8Telethon Kids Institute, 9King Edward Memorial Hospital, 10University of Western Australia

Here, we present a feasibility study to assess a portable amplitude-integrated electroencephalogram (aEEG) recording system during the transport of infants with suspected hypoxic ischemic encephalopathy (HIE).

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