University of Bristol

22 articles published in JoVE

• 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 Mustafa^{4,5 1}Department 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. Banks^{1 1}School 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. Pepple^{1 1}Department 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, Lucy Troman², Franck Duong Van Hoa^{1 1}Department 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 Ciurkot^1,2, Brenda Vonk¹, Thomas E. Gorochowski^3,4, Johannes A. Roubos¹, René Verwaal^{1 1}DSM 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 Boulianne^{1 1}Department 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 Stevens^1,2,3, Sebastian Oltean^{1,2,3 1}Institute 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 Weavers^1,2, Anna Franz¹, Will Wood³, Paul Martin^{1,4 1}School 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. Wallace^{1 1}Division 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. Kauppinen^{1 1}School 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. Quarini^{1 1}Department 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. Adams^{1 1}School 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 Okuda^3,4, Annela M. Seddon¹, Adam W. Perriman⁵, Walther Schwarzacher^{6 1}Bristol 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. Hammond^{1 1}Physiology, 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. Pownall^{5 1}Department 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. Bass^{2 1}School 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. Hodge^{1 1}Physiology 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. Race^{1 1}School 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. Bass^{1 1}School 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. Henley^{1 1}MRC 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 Virji^{1 1}Department 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 Gaston^{1 1}Department 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.