University College London View Institution's Website 67 articles published in JoVE Neuroscience Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals Rhys Burrows1, Chi-Hsuan Ma1, Yujiao Jennifer Sun1 1Institute of Ophthalmology, University College London When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals. Biology Cell-Based Drug Screening for Inhibitors of Autophagy Related 4B Cysteine Peptidase Denise R. B. Pilger1, Christin Luft1, Robin Ketteler1,2 1Laboratory for Molecular Cell Biology, University College London, 2Department of Human Medicine, Medical School Berlin Here, we describe a detailed protocol for the use of a luciferase-based reporter assay in a semi-automated, high-throughput screening format. Biology Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy Gary Hong Chun Chung1, Paul Gissen1,2, Christopher J. Stefan1, Jemima J. Burden1 1MRC Laboratory for Molecular Cell Biology, University College London, 2NIHR Great Ormond Street Hospital Biomedical Research Centre, University College London A simple and comprehensive protocol to acquire three-dimensional details of membrane contact sites between organelles in hepatocytes from the liver or cells in other tissues. Developmental Biology Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System Olivia L. Hagen1, Yehyun Kim1, Elaine Kushkowski1,2, Hannah Rouse3, Kara L. Cerveny1 1Department of Biology, Reed College, 2Committee on Development, Regeneration, and Stem Cell Biology, University of Chicago, 3Department of Cell and Developmental Biology, University College London The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging. Immunology and Infection Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model Chantelle E Bowers1, Virginia L Calder1, John Greenwood1, Malihe Eskandarpour1 1UCL Institute of Ophthalmology, University College London In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts. Neuroscience Expanding the Toolkit for In Vivo Imaging of Axonal Transport Andrew P. Tosolini1,2, David Villarroel-Campos1,2,3, Giampietro Schiavo1,2,3, James N. Sleigh1,2,3 1Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, 2UCL Queen Square Motor Neuron Disease Centre, University College London, 3UK Dementia Research Institute, University College London Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals. Bioengineering A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects Prasad Sawadkar1,3, Jason Wong2, Vivek Mudera1 1Division of Surgery and Interventional Science, University College London, 2Blond McIndoe Laboratories, Division of Cell Matrix and Regenerative Medicine, MAHSC, University of Manchester, 3Department of Surgical Research, Northwick Park Institute for Medical Research, Northwick Park Hospital In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue. Biochemistry Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry Danye Qiu1, Verena B. Eisenbeis1, Adolfo Saiardi2, Henning J. Jessen1 1Institute of Organic Chemistry, University of Freiburg, 2Medical Research Council, Laboratory for Molecular Cell Biology, University College London A procedure for capillary electrophoresis electrospray ionization mass spectrometry for the absolute quantitation of inositol pyrophosphates from mammalian cell extracts is described. Chemistry Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril Weng-I Katherine Chio1, Gemma Davison1, Tabitha Jones1, Jia Liu1, Ivan P. Parkin1, Tung-Chun Lee1,2 1Department of Chemistry, University College London (UCL), 2Institute for Materials Discovery, University College London (UCL) A host-guest complex of cucurbit[7]uril and uric acid was formed in an aqueous solution before adding a small amount into Au NP solution for quantitative surface-enhanced Raman spectroscopy (SERS) sensing using a modular spectrometer. Immunology and Infection Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43 Vanessa S. Terra1, Charles D. Plumptre2, Emma C. Wall3, Jeremy S. Brown2, Brendan W. Wren1 1Faculty of Infections and Tropical Diseases, Department of Infection Biology, London School of Hygiene and Tropical Medicine, 2Division of Medicine, University College London, 3Division of Infection and Immunity, University College London UCL Here, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made. Bioengineering Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning Eleanor C. Mackle*1,2, Jonathan Shapey*1,2,3,4, Efthymios Maneas1,2, Shakeel R. Saeed3,5,6, Robert Bradford3, Sebastien Ourselin4, Tom Vercauteren4, Adrien E. Desjardins1,2 1Wellcome / EPSRC Centre for Interventional and Surgical Sciences, University College London, 2Department of Medical Physics and Biomedical Engineering, University College London, 3Department of Neurosurgery, National Hospital for Neurology and Neurosurgery, 4 This protocol describes the fabrication of a patient specific skull, brain and tumor phantom. It uses 3D printing to create molds, and polyvinyl alcohol (PVA-c) is used as the tissue mimicking material. Neuroscience An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons Nicolás Stuardo1,2, Guillermo Moya-Alvarado1,3, Carolina Ramírez1,3, Giampietro Schiavo4, Francisca C. Bronfman1,3 1Department of Physiology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, 2Department of Molecular and Cellular Biology, Faculty of Biological Sciences, Center for Aging and Regeneration (CARE UC), Pontificia Universidad Católica de Chile, 3Institute of Biomedical Sciences. Faculty of Medicine and Faculty of Life Sciences, Universidad Andrés Bello, 4Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology and UK Dementia Research Institute at UCL, University College London Campus Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a cost-effective manner and is purified by affinity chromatography. BDNFavi is then directly mono-biotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF. Environment Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica Aki Masunaga1, Andrew W. Liu1, Yongkai Tan1, Andrew Scott1, Nicholas M. Luscombe1,2,3 1Okinawa Institute of Science and Technology Graduate University, 2Francis Crick Institute, 3UCL Genetics Institute, Department of Genetics, Evolution and Environment, University College London Oikopleura dioica is a tunicate model organism in various fields of biology. We describe sampling methods, species identification, culturing setup, and culturing protocols for the animals and algal feed. We highlight key factors that helped strengthen the culture system and discuss the possible problems and resolutions. Behavior Implementation of a Real-Time Psychosis Risk Detection and Alerting System Based on Electronic Health Records using CogStack Tao Wang1, Dominic Oliver2, Yamiko Msosa1, Craig Colling3, Giulia Spada2, Łukasz Roguski4, Amos Folarin1, Robert Stewart3,5, Angus Roberts1,3, Richard J. B. Dobson1,3,4,6, Paolo Fusar-Poli2,3,7,8 1 We demonstrate how to deploy a real-time psychosis risk calculation and alerting system based on CogStack, an information retrieval and extraction platform for electronic health records. Medicine Use of Magnetic Resonance Imaging and Biopsy Data to Guide Sampling Procedures for Prostate Cancer Biobanking Susan Heavey1, Aiman Haider2, Ashwin Sridhar3, Hayley Pye1, Greg Shaw1, Alex Freeman2, Hayley Whitaker1 1Molecular Diagnostics and Therapeutics Group, University College London, 2Research Department of Pathology, University College London, 3Department of Uro-Oncology, UCLH NHS Foundation Trust This method involves utilization of clinical diagnostic data for prostate cancer patients in order to guide sampling procedures, when biobanking tissue following radical prostatectomy. This overcomes issues with previously published methods around efficiency and availability of fresh tissue for a wider range of downstream applications. Behavior Application of 3D Printing in the Construction of Burr Hole Ring for Deep Brain Stimulation Implants Jiazhi Chen*1,2, Xinyu Chen*3, Siyuan Lv2, Yuzhen Zhang1, Hao Long1, Kaijun Yang1, Songtao Qi1, Wangming Zhang2, Jun Wang1 1Department of Neurosurgery, Nanfang Hospital, Southern Medical University, 2Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, 3Faculty of Brain Sciences, University of College London Here, we present a protocol to demonstrate 3D printing in the construction of deep brain stimulation implants. Engineering Scalable Quantum Integrated Circuits on Superconducting Two-Dimensional Electron Gas Platform Kaveh Delfanazari1,2, Pengcheng Ma2, Reuben Puddy2, Teng Yi2, Moda Cao2, Yilmaz Gul3, Carly L. Richardson4, Ian Farrer2,5, David Ritchie2, Hannah J. Joyce1, Michael J. Kelly1,2, Charles G. Smith2 1Centre for Advanced Photonics and Electronics, Engineering Department, University of Cambridge, 2Department of Physics, Cavendish Laboratory, University of Cambridge, 3Department of Electronic and Electrical Engineering, University College London, 4Department of Materials Science and Metallurgy, University of Cambridge, 5Department of Electronic and Electrical Engineering, University of Sheffield Quantum integrated circuits (QICs) consisting of array of planar and ballistic Josephson junctions (JJs) based on In0.75Ga0.25As two-dimensional electron gas (2DEG) is demonstrated. Two different methods for fabrication of the two-dimensional (2D) JJs and QICs are discussed followed by the demonstration of quantum transport measurements in sub-Kelvin temperatures. Developmental Biology Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells Balta Lucendo-Villarin1, Hassan Rashidi1,2, Sharmin Alhaque1,3, Lena Fischer1,4, Jose Meseguer-Ripolles1, Yu Wang1, Cliona O'Farrelly4, Michael Themis3, David C. Hay1 1MRC Centre for Regenerative Medicine, University of Edinburgh, 2UCL Great Ormond Street Institute of Child Health, University College London, 3Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, 4School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application. Genetics Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria Peggy Paschke1, David A. Knecht2, Thomas D. Williams1, Peter A. Thomason3, Robert H. Insall3, Jonathan R. Chubb4,5, Robert R. Kay1, Douwe M. Veltman1 1MRC Laboratory of Molecular Biology, 2Department of Molecular and Cell Biology, University of Connecticut, 3Cancer Research UK Beatson Institute Glasgow, 4MRC Laboratory for Molecular Cell Biology, University College London, 5Department of Cell and Developmental Biology, University College London Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media. Neuroscience Biocytin Recovery and 3D Reconstructions of Filled Hippocampal CA2 Interneurons Georgia Economides*1, Svenja Falk*1, Audrey Mercer1 1Department of Pharmacology, University College London The protocol outlined here describes the immunofluorescence analysis, biocytin recovery and high-quality reconstructions of hippocampal CA2 interneurons following the intracellular electrophysiological recordings in vitro, allowing neuronal characterization and ultimately fine neuronal anatomy to be studied. Genetics Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization Fani Memi1, Daniela Tirziu2, Irinna Papangeli3 1Department of Cell and Developmental Biology, University College London, 2Yale Cardiovascular Research Group, Section of Cardiovascular Medicine, Yale School of Medicine, 3Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Yale School of Medicine micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections. Engineering Three-Dimensional Ultrasonic Needle Tip Tracking with a Fiber-Optic Ultrasound Receiver Wenfeng Xia1,2, Simeon J. West3, Malcolm C. Finlay2,4, Rosalind Pratt5,6, Sunish Mathews1,2, Jean-Martial Mari7, Sebastien Ourselin1,2,6, Anna L. David1,5,8,9, Adrien E. Desjardins1,2 1Wellcome/EPSRC Centre for Interventional and Surgical Sciences, University College London, 2Department of Medical Physics and Biomedical Engineering, University College London, 3Department of Anaesthesia, University College Hospital, 4St Bartholomew's Hospital and Queen Mary University of London, 5Institute for Women's Health, University College London, 6Centre for Medical Imaging Computing, University College London, 7GePaSud, University of French Polynesia, 8Department of Development and Regeneration, KU Leuven (Katholieke Universiteit), 9NIHR University College London Hospitals Biomedical Research Centre Accurate and efficient visualization of invasive medical devices is extremely important in many ultrasound-guided minimally invasive procedures. Here, a method for localizing the spatial position of a needle tip relative to the ultrasound imaging probe is presented. Immunology and Infection A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo Line L. Holm1,2,3, Milica Vukmanovic-Stejic4, Thomas Blauenfeldt1, Thomas Benfield2,3, Peter Andersen1, Arne N. Akbar4, Morten Ruhwald1 1Department of Infectious Disease Immunology, Center for Vaccine Research, Statens Serum Institut, 2Department of Infectious Diseases, Hvidovre Hospital, 3Institute of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, 4Division of Infection and Immunity, University College London Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development. Biology Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay James T. Brash1, Christiana Ruhrberg1, Alessandro Fantin1 1UCL Institute of Ophthalmology, University College London Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability. Environment Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction Cecilia S.N. Cheung1,2, Donald J. Weidner1, Li Li1, Philip G. Meredith3, Haiyan Chen1, Matthew Whitaker1, Xianyin Chen4 1Mineral Physics Institute, Department of Geoscience, Stony Brook University, 2Geological Engineering, Department of Civil and Environmental Engineering, University of Wisconsin-Madison, 3Rock and Ice Physics Laboratory, Department of Earth Sciences, University College London, 4Department of Chemistry, Stony Brook University We report detailed procedures for compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus coupled with synchrotron X-radiation. Such experiments allow quantification of the stress distribution within samples, that ultimately sheds light on compaction processes in geomaterials. Neuroscience A Prediction Error-driven Retrieval Procedure for Destabilizing and Rewriting Maladaptive Reward Memories in Hazardous Drinkers Ravi K. Das1, Grace Gale1, Vanessa Hennessy1, Sunjeev K. Kamboj1 1Clinical Psychopharmacology Unit, University College London This manuscript describes a memory retrieval procedure for destabilizing robust reward memories and rewriting them with counterconditioning prior to their reconsolidation. Neuroscience Evaluation of Synapse Density in Hippocampal Rodent Brain Slices Faye McLeod*1, Aude Marzo*1, Marina Podpolny1, Soledad Galli1, Patricia Salinas1 1Department of Cell and Developmental Biology, University College London A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article. Bioengineering The Bionic Clicker Mark I & II Elliott G. Magee1, S. Ourselin2, Daniil Nikitichev2, T. Vercauteren2, Anne Vanhoestenberghe1 1Aspire Create, University College London, 2Translational Imaging Group, CMIC, University College London A device was created to demonstrate electromyography-based control to a lay audience. After the success of the initial device, a second device was made with greater flexibility in functionality for demonstration and research purposes. This protocol describes the process of building and calibrating both devices. Engineering Electrospinning of Photocatalytic Electrodes for Dye-sensitized Solar Cells Nicolò Canever1, Fraser Hughson1, Thomas J. Macdonald2, Thomas Nann1 1The MacDiarmid Institute for Advanced Materials & Nanotechnology, School of, Victoria University of Wellington, 2Department of Chemistry, University College London The overall goal of this project was to use electrospinning to fabricate a photoanode with improved performance for dye-sensitized solar cells. Biology Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper Robert D. M. Gray1,2, Jason Mercer1, Ricardo Henriques1,3 1MRC Laboratory for Molecular Cell Biology, University College London, 2Centre for Mathematics and Physics in Life Sciences and Experimental Biology (CoMPLEX), University College London, 3Department of Cell and Developmental Biology, University College London This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure. Immunology and Infection Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator Juliet R. Foote1, Adam P. Levine1, Philippe Behe1, Michael R. Duchen2, Anthony W. Segal1 1Centre for Molecular Medicine, Division of Medicine, University College London, 2Cell and Developmental Biology, University College London This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis. Bioengineering Medical-grade Sterilizable Target for Fluid-immersed Fetoscope Optical Distortion Calibration Daniil I. Nikitichev*1, Dzhoshkun I. Shakir*1, François Chadebecq1, Marcel Tella1, Jan Deprest1,2, Danail Stoyanov3, Sébastien Ourselin1, Tom Vercauteren1 1Translational Imaging Group, CMIC, University College London, 2Department of Obstetrics and Gynecology, University Hospitals Leuven, 3Surgical Robot Vision Group, CMIC, University College London This article describes the design and development of a sterilizable custom camera optical distortion calibration target for the peri-operative, fluid-immersed calibration of endoscopes during endoscopic interventions. Biology Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy Matthew McKenzie1,2, Sze C. Lim1, Michael R. Duchen3 1Centre for Genetic Diseases, Hudson Institute of Medical Research, 2The Department of Molecular and Translational Sciences, Monash University, 3Department of Physiology, University College London Mitochondria can utilize the electrochemical potential across their inner membrane (ΔΨm) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and ΔΨm in live cells using fluorescent dyes and confocal microscopy. Bioengineering Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing Michelle Griffin1, Yaami Premakumar2, Alexander Seifalian1, Peter Edward Butler1,3, Matthew Szarko2 1Division of Surgery & Interventional Science, University College London (UCL), 2 Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue. Developmental Biology Isolation and Expansion of Adult Canine Hippocampal Neural Precursors Thomas Duncan1, Aileen Lowe1, Marshall A. Dalton2, Michael Valenzuela1 1Regenerative Neuroscience Group, University of Sydney, 2Wellcome Trust Centre for Neuroimaging, Institute of Neurology, University College London The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue. Medicine A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status Wendy E. Heywood*1, Anna Baud*1, Emily Bliss1, Ernestas Sirka1, Jonathan M. Schott2, Henrik Zetterberg3, Daniela Galimberti4, Neil J. Sebire5, Kevin Mills1 1Centre for Translational Omics, Genetics and Genomic Medicine Deptartment, Great Ormond Street Institute of Child Health, University College London, 2Dementia Research Centre, Institute of Neurology, University College London, 3Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy, University of Gothenburg, 4Neurology Unit, Department of Pathophysiology and Transplantation, University of Milan, 5Great Ormond Street Hospital for Children, University College London We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression. Behavior The 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer's Disease Dennis Chan1, Laura Marie Gallaher2, Kuven Moodley2, Ludovico Minati3, Neil Burgess4, Tom Hartley5 1Department of Clinical Neurosciences, University of Cambridge, 2Clinical Imaging Sciences Centre, Brighton and Sussex Medical School, 3U.O. Direzione Scientifica, Fondazione IRCCS Istituto Neurologico Carlo Besta, 4Institute of Cognitive Neuroscience, University College London, 5Department of Psychology, University of York This article describes the 4 Mountains Test (4MT), a hippocampus-dependent test of working allocentric spatial memory. The hippocampus is affected early in Alzheimer's disease (AD) and this article outlines the 4MT methodology and results of patient testing, which demonstrates the value of the 4MT in the diagnosis of pre-dementia AD. 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. Parkin3 1Department 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. Bioengineering Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture Michael P. Storm1, Ian Sorrell2, Rebecca Shipley3, Sophie Regan2, Kim A. Luetchford1, Jean Sathish2, Steven Webb4, Marianne J. Ellis1 1Department of Chemical Engineering and Centre for Regenerative Medicine, University of Bath, 2MRC Centre for Drug Safety Science and Institute of Translational Medicine, University of Liverpool, 3Mechanical Engineering, University College London, 4Department of Applied Mathematics, Liverpool John Moores University The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture. Medicine A Pipeline for 3D Multimodality Image Integration and Computer-assisted Planning in Epilepsy Surgery Mark Nowell1, Roman Rodionov1, Gergely Zombori2, Rachel Sparks2, Michele Rizzi1, Sebastien Ourselin2, Anna Miserocchi3, Andrew McEvoy3, John Duncan1 1Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, 2Center of Medical Imaging and Computing, UCL, 3Department of Neurosurgery, National Hospital for Neurology and Neurosurgery We describe the steps to use our custom designed software for image integration, visualization and planning in epilepsy surgery. Medicine Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation James N. Fullerton1, Elisabetta Segre1, Roel P.H. De Maeyer1, Alexander A.N. Maini1, Derek W. Gilroy1 1Centre for Clinical Pharmacology, Division of Medicine, University College London Intravenous administration of endotoxin reliably elicits dose-dependent physiological and immunological alterations consistent with several pathological states. This reductionist approach permits the investigation, modeling and experimental modification of systemic inflammation and its downstream effects in man. Engineering In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart Sindhu Ramesh*1, Anamika Singh*1, Dasan M. Cibi1, Derek J. Hausenloy1,2,3, Manvendra K. Singh1,2 1Program in Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, 2National Heart Research Institute Singapore, National Heart Centre Singapore, 3The Hatter Cardiovascular Insititute, University College London The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells. Neuroscience Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections Sarah Jolly*1, Alexander Fudge*1, Nigel Pringle1, William D. Richardson1, Huiliang Li1 1Wolfson Institute for Biomedical Research, University College London Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining. Behavior Using Fiberless, Wearable fNIRS to Monitor Brain Activity in Real-world Cognitive Tasks Paola Pinti1,2, Clarisse Aichelburg3, Frida Lind3, Sarah Power1, Elizabeth Swingler3, Arcangelo Merla2, Antonia Hamilton3, Sam Gilbert3, Paul Burgess3, Ilias Tachtsidis1 1Department of Medical Physics and Biomedical Engineering, Malet Place Engineering Building, University College London, 2Infrared Imaging Lab, Institute for Advanced Biomedical Technology (ITAB), Department of Neuroscience, Imaging and Clinical Sciences, University of Chieti-Pescara, 3Institute of Cognitive Neuroscience, Alexandra House, University College London Monitoring brain activity outside the lab without physical constraints presents methodological challenges. A fiberless, wearable functional Near Infrared Spectroscopy (fNIRS) system was used to measure brain activity during an ecological prospective memory task. It was demonstrated that this system could be used to monitor brain activity during non-lab based experiments. Neuroscience Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester Joseph Jebelli*1, Thomas Piers*2, Jennifer Pocock3 1Department of Neurology, University of Washington, 2Therapeutic Innovation Group, School of Life and Medical Sciences, University College London, 3Department of Neuroinflammation, University College London Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature. Biology Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay Sonia Christou-Savina1, Philip L. Beales1, Daniel P. S. Osborn2 1Genetics and Genomic Medicine, Institute of Child Health, University College London, 2 The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD. Developmental Biology Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin Thomas Ramezani1, Derek W. Laux1, Isabel R. Bravo1, Masazumi Tada2, Yi Feng1 1MRC Centre for Inflammation Research, Queen's Medical Research Institute, The University of Edinburgh, 2Department of Cell & Developmental Biology, University College London Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae. Neuroscience Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors Laura E. Brown1, Celine Fuchs1, Martin W. Nicholson1, F. Anne Stephenson1, Alex M. Thomson1, Jasmina N. Jovanovic1 1UCL School of Pharmacy, University College London The molecular mechanisms that co-ordinate the formation of inhibitory GABAergic synapses during ontogeny are largely unknown. To study these processes,we have developed a co-culture model system which incorporates embryonic medium spiny GABAergic neurons cultured together with stably transfected human embryonic kidney 293 (HEK293) cells expressing functional GABAA receptors. Immunology and Infection Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat Fatma Dalgakiran1, Luci A. Witcomb1, Alex J. McCarthy1, George M. H. Birchenough2, Peter W. Taylor1 1School of Pharmacy, University College London, 2Mucin Biology Group, University of Gothenburg Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus. Behavior Transcranial Magnetic Stimulation for Investigating Causal Brain-behavioral Relationships and their Time Course Magdalena W. Sliwinska1, Sylvia Vitello1, Joseph T. Devlin1 1Cognitive, Perceptual & Brain Sciences, University College London Transcranial magnetic stimulation (TMS) is a technique for non-invasively disrupting neural information processing and measuring its effect on behavior. When TMS interferes with a task, it indicates that the stimulated brain region is necessary for normal task performance, allowing one to systematically relate brain regions to cognitive functions. Behavior Cortical Source Analysis of High-Density EEG Recordings in Children Joe Bathelt1, Helen O'Reilly2, Michelle de Haan1 1Cognitive Neuroscience & Neuropsychiatry Section, UCL Institute of Child Health, 2Academic Division of Neonatology, Institute for Women's Health, University College London In recent years, there has been increasing interest in estimating the cortical sources of scalp measured electrical activity for cognitive neuroscience experiments. This article describes how high density EEG is acquired and how recordings are processed for cortical source estimation in children from the age of 2 years at the London Baby Lab. Bioengineering Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder Koon-Yang Lee1,2, Siti Rosminah Shamsuddin3, Marta Fortea-Verdejo1, Alexander Bismarck1,3 1Polymer 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. Neuroscience Preparation of Neuronal Co-cultures with Single Cell Precision Ngoc-Duy Dinh1, Ya-Yu Chiang1,2, Heike Hardelauf1, Sarah Waide1, Dirk Janasek1, Jonathan West1,3 1Leibniz-Institut für Analytische Wissenschaften, ISAS, 2Department of Biochemical Engineering, University College London, 3Institute for Life Sciences, University of Southampton Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs. Neuroscience Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration Miguel Tillo1, Quenten Schwarz1,2, Christiana Ruhrberg1 1UCL Institute of Ophthalmology, University College London, 2Department of Human Immunology, Centre for Cancer Biology, South Australia Embryonic neurons are born in the ventricular zone of the neural tube, but migrate to reach appropriate targets. Facial branchiomotor (FBM) neurons are a useful model to study neuronal migration. This protocol describes the wholemount ex vivo culture of mouse embryo hindbrains to investigate mechanisms that regulate FBM migration. Bioengineering Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration Mattia F. M. Gerli*1, Sara M. Maffioletti*1, Queensta Millet1, Francesco Saverio Tedesco1,2 1Department of Cell and Developmental Biology, University College London, 2Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Hospital Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration. Biology In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors Açelya Yilmazer1, Irene de Lázaro1, Cyrill Bussy1, Kostas Kostarelos2 1Nanomedicine Lab, UCL School of Pharmacy, University College London, 2Nanomedicine Lab, Faculty of Medical & Human Sciences, University of Manchester This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency. Engineering Setting Limits on Supersymmetry Using Simplified Models Christian Gütschow1, Zachary Marshall2,3 1Department of Physics and Astronomy, University College London, 2CERN, 3Physics Division, Lawrence Berkeley National Laboratories This paper demonstrates a protocol for recasting experimental simplified model limits into conservative and aggressive limits on an arbitrary new physics model. Publicly available LHC experimental results can be recast in this manner into limits on almost any new physics model with a supersymmetry-like signature. Immunology and Infection Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection Joseph W. Ndieyira1, Moyu Watari1, Rachel A. McKendry1 1London Centre for Nanotechnology and Departments of Medicine, University College London Acquired resistance to antibiotics is a major public healthcare problem and is presently ranked by the WHO as one of the greatest threats to human life. Here we describe the use of cantilever technology to quantify antibacterial resistance, critical to the discovery of novel and powerful agents against multidrug resistant bacteria. Bioengineering A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix Panagiotis Maghsoudlou*1, Giorgia Totonelli*1, Stavros P Loukogeorgakis1, Simon Eaton1, Paolo De Coppi1 1Surgery Unit, Institute of Child Health and Great Ormond Street Hospital, University College London The article describes a methodology for the production of an acellular matrix from rat intestine. The derivation of intestinal scaffolds is important for future applications in tissue engineering, stem cell biology and drug testing. Neuroscience Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo Simon Tual-Chalot1, Kathleen R. Allinson1, Marcus Fruttiger2, Helen M. Arthur1 1Institute of Genetic Medicine, Newcastle University, 2UCL Institute of Ophthalmology, University College, London The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies. Neuroscience Chicken Embryo Spinal Cord Slice Culture Protocol Kristina C. Tubby*1, Dee Norval*1, Stephen R. Price1 1Research Department of Cell and Developmental Biology, University College London Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr. Biology Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression Marta Gomez-Martinez1, Debora Schmitz2, Alexander Hergovich1 1UCL Cancer Institute, 2Friedrich Miescher Institute for Biomedical Research A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies. Biology A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning Jamie Freeman1, Janos Kriston-Vizi1, Brian Seed2, Robin Ketteler1 1MRC LMCB, University College London, 2Center for Computational and Integrative Biology, Massachusetts General Hospital A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells. Medicine Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression Kate Lawrenson1, Barbara Grun2, Simon A. Gayther1 1Department of Preventive Medicine, University of Southern California, 2Institute for Women's Health, University College London We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development. Neuroscience Electrophysiological Measurements and Analysis of Nociception in Human Infants L. Fabrizi*1, A. Worley*2, D. Patten1, S. Holdridge1, L. Cornelissen1, J. Meek3, S. Boyd2, R. Slater1,4 1Neuroscience, Physiology and Pharmacology, University College London, 2Department of Clinical Neurophysiology, Great Ormond Street Hospital, 3Elizabeth Garrett Anderson Obstetric Hospital, University College Hospital, 4Nuffield Department of Anaesthetics, University of Oxford The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system. Biology Preparation of Quality Inositol Pyrophosphates Omar Loss1, Cristina Azevedo1, Zsolt Szijgyarto1, Daniel Bosch1, Adolfo Saiardi1 1Medical Research Council (MRC), Cell Biology Unit and Laboratory for Molecular Cell Biology, University College London Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates. Neuroscience Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster Hrvoje Augustin1, Marcus J. Allen2, Linda Partridge1 1Institute of Healthy Ageing, and GEE, University College London - UCL, 2School of Biosciences, University of Kent The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.
