Imperial College London

65 articles published in JoVE

• Biology

  
  Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins Brieuc Chauvin*¹, Koyomi Nakazawa*¹, Alexandre Beber^1,7, Aurélie Di Cicco¹, Bassam Hajj¹, François Iv², Manos Mavrakis², Gijsje H. Koenderink³, João T. Cabral⁴, Michaël Trichet⁵, Stéphanie Mangenot*⁶, Aurélie Bertin*^{1 1}Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, Sorbonne Université, ²Institut Fresnel, CNRS UMR7249, Aix Marseille Univ, Centrale Marseille, ³Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, ⁴Department of Chemical Engineering, Imperial College London, ⁵Sorbonne Université, CNRS, Institut de Biologie Paris-Seine (IBPS), Service de microscopie électronique (IBPS-SME), ⁶Laboratoire Matière et Systèmes Complexes (MSC), Université Paris Cité, ⁷Institute of Biotechnology, Czech Academy of Sciences, BIOCEV Septins are cytoskeletal proteins. They interact with lipid membranes and can sense but also generate membrane curvature at the micron scale. We describe in this protocol bottom-up in vitro methodologies for analyzing membrane deformations, curvature-sensitive septin binding, and septin filament ultrastructure.

• Neuroscience

  
  Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology Adrien Rapeaux^1,2,3,4, Omaer Syed⁵, Estelle Cuttaz⁵, Christopher A. R. Chapman⁵, Rylie A. Green⁵, Timothy G. Constandinou^{1,2,3,4 1}Department of Electrical and Electronic Engineering, Imperial College London, ²Centre for Bioinspired Technology, Imperial College London, ³Care Research and Technology (CR&T), Imperial College London and the University of Surrey, ⁴Dementia Institute (UK DRI), ⁵Department of Bioengineering, Imperial College London This protocol describes the preparation of rat whole sciatic nerve tissue for ex vivo electrophysiological stimulation and recording in an environmentally-regulated, two-compartment, perfused saline bath.

• Bioengineering

  
  Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level Emily Masters-Clark¹, Amelia J. Clark¹, Claire E. Stanley^{1 1}Department of Bioengineering, Imperial College London Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

• Bioengineering

  
  Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells Masue Marbiah*^1,2, Pavlos Kotidis*^1,3, Roberto Donini^1,4, Itzcóatl A. Gómez⁵, Ioscani Jimenez del Val⁵, Stuart M. Haslam⁴, Karen M. Polizzi^1,2, Cleo Kontoravdi^{1 1}Department of Chemical Engineering, Imperial College London, ²Imperial College Centre for Synthetic Biology, Imperial College London, ³BioPharm Process Research, GlaxoSmithKline Research and Development, ⁴Department of Life Science, Imperial College London, ⁵School of Chemical & Bioprocess Engineering, University College Dublin The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

• Medicine

  
  Therapy Interventions for Upper Limb Amputees Undergoing Selective Nerve Transfers Agnes Sturma^1,2, Laura A. Hruby^1,3, Anna Boesendorfer¹, Clemens Gstoettner¹, Dario Farina², Oskar C. Aszmann^{1,4 1}Clinical Laboratory for Bionic Extremity Reconstruction, Medical University of Vienna, ²Bioengineering Department, Imperial College London, ³Department of Orthopaedics and Trauma Surgery, Medical University of Vienna, ⁴Department of Plastic and Reconstructive Surgery, Medical University of Vienna This work presents a protocol to enhance prosthetic function after selective nerve transfer surgery. Rehabilitation interventions include patient information and selection, support of wound healing, cortical re-activation of sensory-motor areas of the upper limb, training of selective muscle activation, prosthetic handling in daily life, and regular follow-up assessments.

• Bioengineering

  
  A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications Ming Toh^1,2,3,4, Kameshwari Chengan⁵, Tanith Hanson⁵, Paul S. Freemont^1,2,3,4,6,7, Simon J. Moore^{5 1}Centre for Synthetic Biology and Innovation, South Kensington Campus, ²Department of Medicine, South Kensington Campus, ³Section of Structural and Synthetic Biology, Department of Infectious Disease, Imperial College London, ⁴Sir Alexander Fleming Building, South Kensington Campus, ⁵School of Biosciences, Division of Natural Sciences, University of Kent, ⁶UK Dementia Research Institute Care Research and Technology Centre, Imperial College London; Hammersmith Campus, ⁷UK Innovation and Knowledge Centre for Synthetic Biology (SynbiCITE) and the London Biofoundry, Imperial College Translation & Innovation Hub This protocol details an enhanced method for synthesizing high yields of recombinant proteins from a Streptomyces venezuelae cell-free transcription-translation (TX-TL) system.

• Neuroscience

  
  Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) to Determine the Cellular Origin of Radioactive Signal Quentin Amossé^1,2, Kelly Ceyzériat^1,3,4, Stergios Tsartsalis^1,5, Benjamin B. Tournier^1,2, Philippe Millet^{1,2 1}Division of Adult Psychiatry, Department of Psychiatry, University Hospitals of Geneva, ²Department of Psychiatry, University of Geneva, ³Division of Nuclear medicine and Molecular Imaging, University Hospitals of Geneva, ⁴Division of Radiation Oncology, Department of Oncology, University Hospitals of Geneva, ⁵Department of Brain Sciences, Faculty of Medicine, Imperial College London Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) is a powerful tool to study the role of the 18 kDa translocator protein or Serotonin 5HT2A-receptor expression in Alzheimer's Disease at a cellular scale. This protocol describes the ex-vivo application of FACS-RTT in the TgF344-AD rat model.

• Bioengineering

  
  Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies Kuin T. Pang*^1,2, Mean Ghim*¹, Mehwish Arshad¹, Xiaomeng Wang^2,3,4, Peter D. Weinberg^{1 1}Department of Bioengineering, Imperial College London, ²Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), ³Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, ⁴Singapore Eye Research Institute, The Academia This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

• Biochemistry

  
  Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block Natàlia de Martín Garrido¹, Kailash Ramlaul¹, Christopher H. S. Aylett^{1 1}Section of Structural & Synthetic Biology, Department of Infectious Diseases, Faculty of Medicine, Imperial College London, South Kensington Campus Sample preparation for cryo-electron microscopy (cryo-EM) is a significant bottleneck in the structure determination workflow of this method. Here, we provide detailed methods for using an easy-to-use, three-dimensionally printed block for the preparation of support films to stabilize samples for transmission EM studies.

• Environment

  
  Flash Infrared Annealing for Perovskite Solar Cell Processing Pui Sha Victoria Ling^1,2, Anders Hagfeldt², Sandy Sanchez^{2 1}Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, ²Laboratory of Photomolecular Science (LSPM), École Polytechnique Fédérale de Lausanne (EPFL) We describe a flash infrared annealing method used for the synthesis of perovskite and mesoscopic-TiO2 films. Annealing parameters are varied and optimized for processing on fluorine-doped tin oxide (FTO) glass and indium tin oxide-coated polyethylene terephthalate (ITO PET), subsequently giving devices power conversion efficiencies >20%.

• Cancer Research

  
  Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma Greta Donisi*¹, Marialuisa Barbagallo*², Giovanni Capretti^1,3, Gennaro Nappo^1,3, Panteleimon G. Takis^4,5,6, Alessandro Zerbi^1,3, Federica Marchesi^2,7, Nina Cortese^{2 1}Section of Pancreatic Surgery, Humanitas Clinical and Research Center-IRCCS, ²Department of Immunology and Inflammation, Humanitas Clinical and Research Center-IRCCS, ³Department of Biomedical Sciences, Humanitas University, ⁴Giotto Biotech S.R.L., ⁵Section of Bioanalytical Chemistry, Division of Systems Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College London, ⁶National Phenome Centre, Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, ⁷Department of Medical Biotechnology and Translational Medicine, University of Milan Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

• Cancer Research

  
  Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity Sarah Spear^1,2, Iain A McNeish¹, Melania Capasso^{2,3 1}Division of Cancer, Department of Surgery and Cancer, Imperial College London, ²Centre for Cancer and Inflammation, Barts Cancer Institute, Queen Mary University of London, ³German Center for Neurodegenerative Diseases (DZNE) This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

• Developmental Biology

  
  Cell-cell Fusion of Genome Edited Cell Lines for Perturbation of Cellular Structure and Function Robert Mahen^1,2, Reiner Schulte^{3 1}Photonics Group, Department of Physics, Imperial College London, ²The Medical Research Council Cancer Unit, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, University of Cambridge, ³Cambridge Institute for Medical Research, Cambridge Biomedical Campus, University of Cambridge The purpose of this protocol is to fuse two different cell types to create hybrid cells. Fluorescence microscopy analysis of fused cells is used to track the cell of origin of cellular organelles. This assay can be used to explore how cellular structure and function respond to perturbation by cell fusion.

• Neuroscience

  
  Surface Electromyographic Biofeedback as a Rehabilitation Tool for Patients with Global Brachial Plexus Injury Receiving Bionic Reconstruction Laura A. Hruby^1,2, Agnes Sturma^1,3, Oskar C. Aszmann^{1,4 1}Clinical Laboratory for Bionic Extremity Reconstruction, Medical University of Vienna, ²Department of Orthopaedics and Trauma Surgery, Medical University of Vienna, ³Department of Bioengineering, Imperial College London, ⁴Division of Plastic and Reconstructive Surgery, Department of Surgery, Medical University of Vienna Optimal functional outcomes after bionic reconstruction in patients with global brachial plexus injury depend on a structured rehabilitation protocol. Surface electromyographic guided training may improve the amplitude, separation and consistency of EMG signals, which - after elective amputation of a functionless hand - control and drive a prosthetic hand.

• Medicine

  
  Structured Motor Rehabilitation After Selective Nerve Transfers Agnes Sturma^1,2,3, Laura A. Hruby^1,4, Dario Farina², Oskar C. Aszmann^{1,5 1}Clinical Laboratory for Bionic Extremity Reconstruction, Medical University of Vienna, ²Bioengineering Department, Imperial College London, ³Master's Degree Program Health Assisting Engineering, University of Applied Sciences FH Campus Wien, ⁴Department of Orthopedics and Trauma Surgery, Medical University of Vienna, ⁵Division of Plastic and Reconstructive Surgery, Department of Surgery, Medical University of Vienna Here, we present a protocol for the motor rehabilitation of patients with severe nerve injuries and selective nerve transfer surgery. It aims at restoring the motor function proposing several stages in patient education, early-stage therapy after surgery and interventions for rehabilitation after successful re-innervation of the nerve’s target.

• Immunology and Infection

  
  Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex Masanori Asai*¹, Yanwen Li*¹, Jasmeet Singh Khara^1,2, Camilla A. Gladstone¹, Brian D. Robertson³, Paul R. Langford*¹, Sandra M. Newton*^{1 1}Section of Pediatric Infectious Diseases and Allergy, Department of Medicine, Imperial College London, ²Department of Pharmacy, National University of Singapore, ³MRC Centre for Molecular Bacteriology and Infection, Department of Medicine, Imperial College London Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

• Genetics

  
  Transcription Start Site Mapping Using Super-low Input Carrier-CAGE Nevena Cvetesic^1,2, Elena Pahita^1,2, Boris Lenhard^{1,2,3 1}Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, ²MRC London Institute of Medical Sciences, ³Sars International Centre for Marine Molecular Biology, University of Bergen Cap Analysis of Gene Expression (CAGE) is a method for genome-wide quantitative mapping of mRNA 5’ends to capture RNA polymerase II transcription start sites at a single-nucleotide resolution. This work describes a low-input (SLIC-CAGE) protocol for generation of high-quality libraries using nanogram-amounts of total RNA.

• Immunology and Infection

  
  Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation Xiang Zhao¹, Maryam Hamidinia¹, Joanna Ai Ling Choo¹, Chien Tei Too^1,2, Zi Zong Ho³, Ee Chee Ren⁴, Antonio Bertoletti³, Paul A. MacAry^1,2,5, Keith G. Gould⁶, Joanna Brzostek¹, Nicholas R.J. Gascoigne^{1,2,5 1}Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, ²Immunology Programme, Life Sciences Institute, National University of Singapore, ³Emerging Infectious Diseases Program, Duke-NUS Graduate Medical School, ⁴Singapore Immunology Network, A*STAR, ⁵NUS Graduate School for Integrative Sciences and Engineering (NGS), National University of Singapore, ⁶Department of Immunology, Wright-Fleming Institute, Imperial College London This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

• Immunology and Infection

  
  "Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection Ana Maria Ortega-Prieto¹, Jessica Katy Skelton¹, Catherine Cherry¹, Marco Antonio Briones-Orta¹, Charlotte Alexandra Hateley¹, Marcus Dorner^{1 1}Section of Virology, Department of Medicine, Imperial College The goal of this protocol is to provide a step-by-step guide to perform 3-D "liver-on-a-chip" infection experiments with the hepatitis B virus.

• Neuroscience

  
  A Novel In Vitro Model of Blast Traumatic Brain Injury Rita Campos-Pires^1,2, Amina Yonis¹, Warren Macdonald^2,3, Katie Harris¹, Christopher J. Edge^4,5, Peter F. Mahoney⁶, Robert Dickinson^{1,2 1}Anaesthetics, Pain Medicine and Intensive Care Section, Department of Surgery and Cancer, Imperial College London, ²Royal British Legion Centre for Blast Injury Studies, Department of Bioengineering, Imperial College London, ³Department of Bioengineering, Imperial College London, ⁴Department of Life Sciences, Imperial College London, ⁵Department of Anaesthetics, Royal Berkshire Hospital NHS Foundation Trust, ⁶Royal Centre for Defence Medicine, Medical Directorate Joint Force Command This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

• Engineering

  
  Pore-scale Imaging and Characterization of Hydrocarbon Reservoir Rock Wettability at Subsurface Conditions Using X-ray Microtomography Amer M. Alhammadi¹, Ahmed AlRatrout¹, Branko Bijeljic¹, Martin J. Blunt^{1 1}Department of Earth Science and Engineering, Imperial College London This protocol is presented to characterize the complex wetting conditions of an opaque porous medium (hydrocarbon reservoir rock) using three-dimensional images obtained by X-ray microtomography at subsurface conditions.

• Engineering

  
  Film Control to Study Contributions of Waves to Droplet Impact Dynamics on Thin Flowing Liquid Films Idris T. Adebayo¹, Omar K. Matar^{1 1}Department of Chemical Engineering, Imperial College London A protocol to study the contributions of waves to droplet impact dynamics on flowing liquid films is presented.

• Cancer Research

  
  Counting Proteins in Single Cells with Addressable Droplet Microarrays Stelios Chatzimichail*¹, Pashiini Supramaniam*¹, Oscar Ces¹, Ali Salehi-Reyhani^{1 1}Institute of Chemical Biology, Department of Chemistry, Imperial College London Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

• Neuroscience

  
  Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent Sungmin Kang¹, Michael Bruyns-Haylett², Yurie Hayashi¹, Ying Zheng^{1 1}School of Biological Sciences, Whiteknights, University of Reading, ²Department of Bioengineering, Imperial College This protocol describes a simple method for concurrent recording of co-localized electroencephalography (EEG) and multi-laminar local field potential in an anesthetized rat. A burr hole drilled in the skull for the insertion of a microelectrode is shown to produce negligible distortion of the EEG signal.

• Bioengineering

  
  Evaluating Primary Blast Effects In Vitro Niall J. Logan¹, Hari Arora¹, Claire A. Higgins^{1 1}Department of Bioengineering, Imperial College London Understanding how cells are modulated by exposure to shock waves can help identify the mechanisms behind injuries triggered from blast events. This protocol uses custom-built shock tube equipment to apply shock waves at a range of pressures to cell monolayers and to identify the subsequent effects on cell viability.

• Chemistry

  
  Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene Rebecca A. Ryan¹, Sophie Williams¹, Andrew V. Martin¹, Ruben A. Dilanian¹, Connie Darmanin², Corey T. Putkunz¹, David Wood³, Victor A. Streltsov⁴, Michael W.M. Jones⁵, Naylyn Gaffney⁶, Felix Hofmann⁷, Garth J. Williams⁸, Sebastien Boutet⁹, Marc Messerschmidt¹⁰, M. Marvin Seibert¹¹, Evan K. Curwood¹¹, Eugeniu Balaur², Andrew G. Peele⁵, Keith A. Nugent², Harry M. Quiney¹, Brian Abbey^{2 1}ARC 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.

• Environment

  
  Measurement of the Rheology of Crude Oil in Equilibrium with CO₂ at Reservoir Conditions Ruien Hu¹, John Crawshaw^{1 1}Chemical Engineering Department, Imperial College London, Qatar Carbonate and Carbon Storage Research Centre A method for measuring the rheology of crude oil in equilibrium with carbon dioxide at reservoir conditions is presented.

• Engineering

  
  A Novel Biaxial Testing Apparatus for the Determination of Forming Limit under Hot Stamping Conditions Zhutao Shao¹, Nan Li^{1 1}Department of Mechanical Engineering, Imperial College London This protocol proposes a novel biaxial testing system used on a resistance heating uniaxial tensile test machine in order to determine the forming limit diagram (FLD) of sheet metals under hot stamping conditions.

• Engineering

  
  Dynamic Pore-scale Reservoir-condition Imaging of Reaction in Carbonates Using Synchrotron Fast Tomography Hannah P. Menke¹, Matthew G. Andrew², Joan Vila-Comamala³, Christoph Rau³, Martin J. Blunt¹, Branko Bijeljic^{1 1}Department of Earth Science and Engineering, Imperial College London, ²Carl Zeiss X-Ray Microscopy, ³Diamond Manchester Imaging Branchline (I13-2), Diamond Lightsource Synchrotron fast tomography was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 µm resolution over a period of 2 h.

• Biology

  
  Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy Frederik Görlitz*¹, Douglas J. Kelly*¹, Sean C. Warren¹, Dominic Alibhai², Lucien West³, Sunil Kumar¹, Yuriy Alexandrov¹, Ian Munro¹, Edwin Garcia¹, James McGinty¹, Clifford Talbot¹, Remigiusz A. Serwa⁴, Emmanuelle Thinon⁴, Vincenzo da Paola³, Edward J. Murray⁵, Frank Stuhmeier⁶, Mark A. A. Neil¹, Edward W. Tate⁴, Christopher Dunsby^1,7, Paul M. W. French^{1 1}Photonics Group, Department of Physics, Imperial College London, ²Institute for Chemical Biology, Department of Chemistry, Imperial College London, ³MRC Clinical Sciences Centre, Hammersmith Hospital, ⁴Chemical Biology Section, Department of Chemistry, Imperial College London, ⁵Retroscreen Virology Ltd, ⁶Pfizer Global Research and Development, Pfizer Limited, Sandwich, Kent, UK, ⁷Centre for Histopathology, Imperial College London We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in µManager and data analysis is undertaken in FLIMfit.

• 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.

• Engineering

  
  Knowledge Based Cloud FE Simulation of Sheet Metal Forming Processes Du Zhou¹, Xi Yuan¹, Haoxiang Gao¹, Ailing Wang¹, Jun Liu¹, Omer El Fakir¹, Denis J. Politis¹, Liliang Wang¹, Jianguo Lin^{1 1}Department of Mechanical Engineering, Imperial College London The following paper presents a novel FE simulation technique (KBC-FE), which reduces computational cost by performing simulations on a cloud computing environment, through the application of individual modules. Moreover, it establishes a seamless collaborative network between world leading scientists, enabling the integration of cutting edge knowledge modules into FE simulations.

• Neuroscience

  
  Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer Chun-Chieh Lin¹, Olena Riabinina², Christopher J. Potter^{1 1}The Solomon H. Snyder Department of Neuroscience, Center for Sensory Biology, Johns Hopkins University School of Medicine, ²MRC Clinical Sciences Center, Imperial College London We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

• Environment

  
  Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids Florence J. V. Gschwend¹, Agnieszka Brandt¹, Clementine L. Chambon¹, Wei-Chien Tu¹, Lisa Weigand¹, Jason P. Hallett^{1 1}Department of Chemical Engineering, Imperial College London The pretreatment of lignocellulosic biomass with protic low-cost ionic liquids is shown, resulting in a delignified cellulose-rich pulp and a purified lignin. The pulp gives rise to high glucose yields after enzymatic saccharification.

• Engineering

  
  Advanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence Imaging Colin R. Crick*¹, Sacha Noimark*^2,3, William J. Peveler*³, Joseph C. Bear³, Aleksandar P. Ivanov¹, Joshua B. Edel¹, Ivan P. Parkin^{3 1}Department of Chemistry, Imperial College London, ²Department of Medical Physics and Biomedical Engineering, University College London, ³Department of Chemistry, University College London Here we present a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of cadmium selenide quantum dots can be accurately visualized through cross-sectional fluorescence imaging.

• Medicine

  
  A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine Mitsuhiro Yoneda¹, Alfredo A. Molinolo², Jerrold M. Ward³, Shioko Kimura¹, Robert A. Goodlad^{4 1}National Cancer Institute, Laboratory of Metabolism, National Institutes of Health, ²National Institute of Dental and Craniofacial Research, Oral and Pharyngeal Cancer Branch, National Institutes of Health, ³Global VetPathology, ⁴Centre for Pathology, Hammersmith Hospital, Imperial College The use of a simple device to cut and ‘roll’ mouse intestines to rapidly prepare whole mount preparations is described.

• Engineering

  
  A Method for Studying the Temperature Dependence of Dynamic Fracture and Fragmentation David R. Jones¹, David J. Chapman¹, Daniel E. Eakins^{1 1}Institute of Shock Physics, Imperial College London Fracture and fragmentation are late stage phenomena in dynamic loading scenarios and are typically studied using explosives. We present a technique for driving expansion using a gas gun which uniquely enables control of both loading rate and sample temperature.

• Chemistry

  
  The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphos^Ph Complexes Andreas Phanopoulos¹, Nicholas Long¹, Philip Miller^{1 1}Department of Chemistry, Imperial College London Ruthenium phosphine complexes are widely used for homogeneous catalytic reactions such as hydrogenations. The synthesis of a series of novel tridentate ruthenium complexes bearing the N-triphos ligand N(CH2PPh2)3 is reported. Additionally, the stoichiometric reaction of a dihydride Ru–N-triphos complex with levulinic acid is described.

• Medicine

  
  Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines Ylenia Lombardo*¹, Alexander de Giorgio*¹, Charles R. Coombes¹, Justin Stebbing¹, Leandro Castellano^{1 1}Department of Surgery and Cancer, Imperial College London Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

• Neuroscience

  
  Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus Tharakeswari Selvakumar¹, Kambiz N. Alavian², Travis Tierney^{1 1} The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

• Engineering

  
  Reservoir Condition Pore-scale Imaging of Multiple Fluid Phases Using X-ray Microtomography Matthew Andrew¹, Branko Bijeljic¹, Martin Blunt^{1 1}Department of Earth Science and Engineering, Imperial College London We present a methodology for the imaging of multiple fluid phases at reservoir conditions by the use of x-ray microtomography. We show some representative results of capillary trapping in a carbonate rock sample.

• Neuroscience

  
  Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains Maria Weinert¹, Tharakeswari Selvakumar², Travis S. Tierney², Kambiz N. Alavian^{1,3 1}Division of Brain Sciences, Department of Medicine, Imperial College London, ² The causes of degeneration of midbrain dopaminergic neurons during Parkinson’s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

• Immunology and Infection

  
  Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions Maria J. Mazon Moya¹, Emma Colucci-Guyon², Serge Mostowy^{1 1}Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, ²Département de Biologie du Développement et des Cellules Souches, Institut Pasteur, Unité Macrophages et Développement de l'Immunité To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

• Biology

  
  In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow Mark K. Scott¹, Olufolake Akinduro², Cristina Lo Celso^{2 1}Department of Life Science and Facility for Imaging by Light Microscopy, Imperial College London, ²Department of Life Sciences, Imperial College London The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

• Bioengineering

  
  Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder Koon-Yang Lee^1,2, Siti Rosminah Shamsuddin³, Marta Fortea-Verdejo¹, Alexander Bismarck^{1,3 1}Polymer and Composite Engineering (PaCE) Group, Institute of Materials Chemistry and Research, University of Vienna, ²Department of Chemical Engineering, University College London, ³Polymer and Composite Engineering (PaCE) Group, Department of Chemical Engineering, Imperial College London We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

• Chemistry

  
  Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography Tom Boultwood¹, Dominic P. Affron¹, James A. Bull^{1 1}Department of Chemistry, Imperial College London A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography.

• Bioengineering

  
  FIBS-enabled Noninvasive Metabolic Profiling Alireza Behjousiar^1,2, Antony Constantinou², Karen M. Polizzi², Cleo Kontoravdi^{1 1}Centre for Process Systems Engineering, Department of Chemical Engineering and Chemical Technology, Imperial College London, ²Centre for Synthetic Biology and Innovation, Division of Molecular Biosciences, Imperial College London A description of how to calibrate Förster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented. The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

• Medicine

  
  Intramyocardial Cell Delivery: Observations in Murine Hearts Tommaso Poggioli^1,2, Padmini Sarathchandra^1,2, Nadia Rosenthal^2,3, Maria P. Santini^{1,2 1}Magdi Yacoub Institute, Imperial College London, ²National Heart and Lung Institute, Imperial College London, ³Australian Regenerative Medicine Institute, Monash University Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

• Immunology and Infection

  
  Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection Clare R. Harding¹, Gunnar N. Schroeder¹, James W. Collins¹, Gad Frankel^{1 1}Center for Molecular Bacteriology and Infection, Imperial College London The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

• Immunology and Infection

  
  4D Multimodality Imaging of Citrobacter rodentium Infections in Mice James William Collins¹, Jeffrey A Meganck², Chaincy Kuo², Kevin P Francis², Gad Frankel^{1 1}MRC Centre for Molecular Bacteriology and Infection, Division of Cell & Molecular Biology, Imperial College London, ²Preclinical Imaging, Caliper- A PerkinElmer Company Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with μCT imaging to create a 4D movie of C. rodentium infection.

• Immunology and Infection

  
  A Visual Assay to Monitor T6SS-mediated Bacterial Competition Abderrahman Hachani¹, Nadine S. Lossi¹, Alain Filloux^{1 1}MRC Centre for Molecular Bacteriology and Infection, Division of Cell and Molecular Biology, Imperial College London We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

• Medicine

  
  Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens Dorothée Sturm^1,2, Lorella Marselli³, Florian Ehehalt^1,2, Daniela Richter¹, Marius Distler², Stephan Kersting^1,2, Robert Grützmann², Krister Bokvist⁴, Philippe Froguel⁵, Robin Liechti⁶, Anne Jörns⁷, Paolo Meda⁸, Gustavo Bruno Baretton⁹, Hans-Detlev Saeger², Anke M. Schulte¹⁰, Piero Marchetti³, Michele Solimena^{1 1}Molecular Diabetology, Paul Langerhans Institute Dresden, ²Department of GI-, Thoracic- and Vascular Surgery, University Hospital Carl Gustav Carus, University of Technology Dresden, ³Department of Endocrinology and Metabolism, Metabolic Unit University of Pisa, ⁴Labs DC0522, Lilly Corporate Center, ⁵Genomics, Faculty of Medicine Imperial College London, ⁶Vital-IT, SIB Swiss Institute of Bioinformatics, ⁷Clinical Biochemistry, Hannover Medical School, ⁸Cell Physiology and Metabolism, Medical School, University of Geneva, ⁹Department of Pathology, University Hospital Carl Gustav Carus, University of Technology Dresden, ¹⁰R&D DIAB Division / Translational Medicine, Sanofi-Aventis Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

• Biology

  
  High-throughput Purification of Affinity-tagged Recombinant Proteins Simone C. Wiesler¹, Robert O.J. Weinzierl^{1 1}Department of Life Sciences, Imperial College London We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

• Immunology and Infection

  
  Reverse Genetics Mediated Recovery of Infectious Murine Norovirus Armando Arias*¹, Luis Ureña*¹, Lucy Thorne¹, Muhammad A. Yunus¹, Ian Goodfellow^{1 1}Section of Virology, Imperial College London Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

• Medicine

  
  Roux-en-Y Gastric Bypass Operation in Rats Marco Bueter^1,2, Kathrin Abegg^2,3, Florian Seyfried⁴, Thomas A. Lutz^2,3, Carel W. le Roux^{4 1}Department of Surgery, University Hospital Zürich, ²Zürich Centre for Integrative Human Physiology, University of Zürich, ³Institute of Veterinary Physiology, Vetsuisse Faculty, University of Zürich, ⁴Imperial Weight Centre, Department of Investigative Medicine, Imperial College London Numerous studies using gastric bypass rat models have been recently conducted to uncover the underlying physiological mechanisms of Roux-en-Y gastric bypass operations. This article aims to demonstrate and discuss the technical and experimental details of our published gastric bypass rat model to understand advantages and limitations of this experimental tool.

• Medicine

  
  The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System Anne Klomp¹, Jordi L. Tremoleda², Anouk Schrantee¹, Willy Gsell², Liesbeth Reneman^{1 1}Department of Radiology, Brain Imaging Center, Academic Medical Center Amsterdam, ²Biological Imaging Centre, MRC Clinical Sciences Centre, Imperial College London The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

• Bioengineering

  
  Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis Teresa Mortera-Blanco¹, Maria Rende¹, Hugo Macedo¹, Serene Farah¹, Alexander Bismarck¹, Athanasios Mantalaris¹, Nicki Panoskaltsis^{2 1}Department of Chemical Engineering and Chemical Technology, South Kensington campus, Imperial College London, ²Department of Hematology, Northwick Park & St. Mark's campus, Imperial College London A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

• Biology

  
  Identification of Protein Interacting Partners Using Tandem Affinity Purification Dalan Bailey*¹, Luis Urena*¹, Lucy Thorne¹, Ian Goodfellow^{1 1}Section of Virology, Department of Medicine, Imperial College London Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

• Bioengineering

  
  Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells Klaus Suhling¹, James A. Levitt¹, Pei- Hua Chung¹, Marina. K. Kuimova², Gokhan Yahioglu^{3 1}Department of Physics, King's College London, ²Department of Chemistry, Imperial College London, ³PhotoBiotics Ltd Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

• Medicine

  
  Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice Michael T. Kuhlmann¹, Simon Cuhlmann^2,3, Irmgard Hoppe¹, Rob Krams³, Paul C. Evans², Gustav J. Strijkers⁴, Klaas Nicolay⁴, Sven Hermann¹, Michael Schäfers^{1 1}European Institute for Molecular Imaging, Westfälische Wilhelms-University Münster, ²British Heart Foundation Cardiovascular Sciences Unit, Imperial College London, ³Department of Bioengineering, Imperial College London, ⁴Biomedical Engineering, Eindhoven University of Technology The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

• Bioengineering

  
  Fluorescence detection methods for microfluidic droplet platforms Xavier Casadevall i Solvas¹, Xize Niu¹, Katherine Leeper¹, Soongwon Cho¹, Soo-Ik Chang², Joshua B. Edel¹, Andrew J. deMello^{3 1}Department of Chemistry, Imperial College London, ²Department of Biochemistry, Protein Chip Research Center, Chungbuk National University, ³Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering, ETH Zurich Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

• Immunology and Infection

  
  A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux) Sandra Newton¹, Adrian Martineau², Beate Kampmann^{1 1}Department of Paediatrics, Imperial College London, ²Centre for Health Sciences, Barts & The London School of Medicine and Dentistry We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). “Survival” of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

• Neuroscience

  
  An Experimental Platform to Study the Closed-loop Performance of Brain-machine Interfaces Naveed Ejaz¹, Kris D. Peterson¹, Holger G. Krapp^{1 1}Department of Bioengineering, Imperial College London We use a closed-loop fly-machine interface to investigate general principles in neuronal control.

• Biology

  
  Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay Efstathia Thymiakou¹, Vasso Episkopou^{1 1}Medical Research Council, Clinical Sciences Centre, Imperial College, Hammersmith Hospital Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

• Biology

  
  The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis Mark F Brady¹, Jorge Coronel², Robert H Gilman³, David AJ Moore^{4 1}The Warren Alpert Medical School of Brown University, ²Laboratorio de Investigacion de Enfermedades Infecciosas, Universidad Peruana Cayetano Heredia, ³Department of International Health, Johns Hopkins Bloomberg School of Public Health, ⁴Wellcome Trust Centre for Clinical Tropical Medicine, Imperial College London The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method.