University of Nottingham View Institution's Website 18 articles published in JoVE Biology Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications Paulina M. Durczak*1, Kathryn L. Fair*1, Nicholas Jinks1, Sara Cuevas Ocaña1, Carlos B. Sainz Zuñiga1, Nicholas R.F. Hannan1 1Biodiscovery Institute, Translational Medical Sciences, School of Medicine, University of Nottingham This protocol allows for the differentiation of human pluripotent cells into intestinal organoids. The protocol mimics normal human development by differentiating cells into a population of definitive endoderm, hindgut endoderm and then intestinal epithelium. This makes the protocol suitable for studying both intestinal development as well as disease modelling applications. Behavior Assessment of Spatial Lingual Tactile Sensitivity using a Gratings Orientation Test Noemi Sofia Rabitti1, Marta Appiani1, Camilla Cattaneo1, Rebecca Ford2, Monica Laureati1 1Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, 2Division of Food, Nutrition, and Dietetics, School of Biosciences, University of Nottingham This work illustrates a standard procedure and threshold determination by the R-index to assess spatial lingual tactile sensitivity using a gratings orientation test. Bioengineering Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells Emma Pipó-Ollé1, Prasad Walke2, Martine K. Notabi1, Rime B. El-Houri1, Morten Østergaard Andersen1, David Needham2,3,4, Eva C. Arnspang1 1Department of Chemical Engineering, Biotechnology and Environmental Technology, University of Southern Denmark, 2Center for Single Particle Science and Engineering (SPSE), Institute for Molecular Medicine, Health Sciences, University of Southern Denmark, 3Department of Mechanical Engineering and Material Science, Duke University, 4School of Pharmacy, University of Nottingham This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique. Medicine Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice Melissa Trotman-Lucas1, Michael E. Kelly2, Justyna Janus2, Claire L. Gibson1,3 1Department of Neuroscience, Psychology and Behaviour, University of Leicester, 2Preclinical Imaging Facility, Core Biotechnology Services, University of Leicester, 3School of Psychology, University of Nottingham Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory. Immunology and Infection A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection Ola H. Negm1,2, Mohamed Hamed1,2, Tanya M. Monaghan3 1Breast Surgery Group, Division of Medical Sciences and Graduate Entry Medicine, School of Medicine, Queen's Medical Centre, University of Nottingham, 2Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, 3Nottingham Digestive Diseases Centre and NIHR Nottingham Digestive Diseases Biomedical Research Centre, School of Medicine, Queen's Medical Centre, University of Nottingham This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin. Genetics Immunostaining for DNA Modifications: Computational Analysis of Confocal Images Ashley H. Ramsawhook*1, Lara C. Lewis*1, Maria Eleftheriou1, Abdulkadir Abakir1, Paulina Durczak1, Robert Markus2, Seema Rajani2, Nicholas R.F. Hannan1, Beth Coyle3, Alexey Ruzov1 1Division of Cancer and Stem Cells, School of Medicine, Centre for Biomolecular Sciences, University of Nottingham, 2School of Life Sciences Imaging (SLIM), School of Life Sciences, University of Nottingham, 3Children's Brain Tumour Research Centre, School of Medicine, QMC, University of Nottingham Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described. Biology Volume Segmentation and Analysis of Biological Materials Using SuRVoS (Super-region Volume Segmentation) Workbench Michele C. Darrow1, Imanol Luengo1,2, Mark Basham1, Matthew C. Spink1, Sarah Irvine1, Andrew P. French2, Alun W. Ashton1, Elizabeth M.H. Duke1 1Science Division, Harwell Science and Innovation Campus, Diamond Light Source, 2School of Computer Science, University of Nottingham Segmentation of three-dimensional data from many imaging techniques is a major bottleneck in analysis of complex biological systems. Here, we describe the use of SuRVoS Workbench to semi-automatically segment volumetric data at various length-scales using example datasets from cryo-electron tomography, cryo soft X-ray tomography, and phase contrast X-ray tomography techniques. Chemistry Microfluidic-based Synthesis of Covalent Organic Frameworks (COFs): A Tool for Continuous Production of COF Fibers and Direct Printing on a Surface Afshin Abrishamkar1, David Rodríguez-San-Miguel2, Jorge Andrés Rodríguez Navarro3, Romen Rodriguez-Trujillo4, David B. Amabilino5, Ruben Mas-Ballesté2, Félix Zamora2,6,7, Andrew J. deMello1, Josep Puigmarti-Luis1 1Institute of Chemical and Bioengineering, Department of Chemistry and Applied Bioscience, ETH Zurich, 2Departamento de Química Inorgánica, Universidad Autónoma de Madrid, 3Departamento de Química Inorgánica, Universidad de Granada, 4Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), 5School of Chemistry, University of Nottingham, 6Condensed Matter Physics Center (IFMAC), Universidad Autónoma de Madrid, 7Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia) We present a novel microfluidic-based method for synthesis of covalent organic frameworks (COFs). We demonstrate how this approach can be used to produce continuous COF fibers, and also 2D or 3D COF structures on surfaces. Engineering Magnetically Induced Rotating Rayleigh-Taylor Instability Matthew M. Scase*1, Kyle A. Baldwin*2, Richard J. A. Hill*3 1School of Mathematical Sciences, University of Nottingham, 2Faculty of Engineering, University of Nottingham, 3School of Physics and Astronomy, University of Nottingham We present a protocol for preparing a two-layer density-stratified liquid that can be spun-up into solid body rotation and subsequently induced into Rayleigh-Taylor instability by applying a gradient magnetic field. Biology Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry Abdulkadir Abakir1, Lee Wheldon2, Andrew D. Johnson3, Patrick Laurent1, Alexey Ruzov4 1Laboratoire de Neurophysiologie (CP601), ULB Neuroscience Institute (UNI), Université Libre de Bruxelles, 2Medical Molecular Sciences, Centre for Biomolecular Sciences, University of Nottingham, 3School of Life Sciences, University of Nottingham, 4Division of Cancer and Stem Cells, Centre for Biomolecular Sciences, School of Medicine, University of Nottingham Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification. Chemistry Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping, Manipulation and Controlled Chemical Treatment Afshin Abrishamkar1,2, Markos Paradinas3, Elena Bailo4, Romen Rodriguez-Trujillo5, Raphael Pfattner5, René M. Rossi1, Carmen Ocal5, Andrew J. deMello2, David B. Amabilino6, Josep Puigmartí-Luis1 1Empa - Swiss Federal Laboratories for Materials Science and Technology, 2Institute of Chemical and Bioengineering, Department of Chemistry and Applied Bioscience, ETH Zurich, 3ICN2-Institut Catala de Nanociencia i Nanotecnologia, 4WITec GmbH, 5Institut de Ciència de Materials de Barcelona, 6School of Chemistry, The University of Nottingham Herein, we describe the fabrication and operation of a double-layer microfluidic system made of polydimethylsiloxane (PDMS). We demonstrate the potential of this device for trapping, directing the coordination pathway of a crystalline molecular material and controlling chemical reactions onto on-chip trapped structures. Developmental Biology Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression Rabeea H. Mohammed1, Dylan Sweetman1 1Division of Animal Sciences, School of Biosciences, University of Nottingham By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction. Bioengineering Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall Jack C. Bridge1, Jonathan W. Aylott2, Christopher E. Brightling5, Amir M. Ghaemmaghami3, Alan J. Knox4, Mark P. Lewis6, Felicity R.A.J. Rose1, Gavin E. Morris1 1Division of Drug Delivery and Tissue Engineering, University of Nottingham, 2Laboratory of Biophysics and Surface Analysis, School of Pharmacy, University of Nottingham, 3Division of Immunology and Allergy, School of Molecular Medical Sciences, University of Nottingham, 4Division of Respiratory Medicine, School of Clinical Sciences, University of Nottingham, 5NIHR Respiratory Biomedical Research Unit, University of Leicester, 6School of Sport, Exercise, and Health Sciences, Loughborough University Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall. Biology Methods to Assess Subcellular Compartments of Muscle in C. elegans Christopher J. Gaffney1, Joseph J. Bass1, Thomas F. Barratt1, Nathaniel J. Szewczyk1 1MRC/ARUK Centre for Musculoskeletal Ageing Research, University of Nottingham Skeletal muscle is essential for locomotion and is the bodies’ main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes. Chemistry Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins Jennifer T. Patel1, Hannah R. Belsham1, Alexandra J. Rathbone1, Claire T. Friel1 1School of Life Sciences, University of Nottingham Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence. Bioengineering Self-reporting Scaffolds for 3-Dimensional Cell Culture Helen Harrington1, Felicity R.A.J. Rose2, Jonathan W. Aylott3, Amir M. Ghaemmaghami1 1School of Molecular Medical Sciences, University of Nottingham, 2Division of Drug Delivery and Tissue Engineering, University of Nottingham, 3Laboratory of Biophysics and Surface Analysis, University of Nottingham Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment. Behavior Best Current Practice for Obtaining High Quality EEG Data During Simultaneous fMRI Karen J. Mullinger1, Pierluigi Castellone2, Richard Bowtell1 1Sir Peter Mansfield Magnetic Resonance Centre, School of Physics and Astronomy, University of Nottingham, 2Brain Products GmbH Simultaneous electroencephalography (EEG) and functional Magnetic Resonance imaging (fMRI) is a powerful neuroimaging tool. However, the inside of an MRI scanner forms a difficult environment for EEG data recording and safety must be considered whenever operating EEG equipment inside a scanner. Here, we present an optimised EEG-fMRI data acquisition protocol. Bioengineering Polymer Microarrays for High Throughput Discovery of Biomaterials Andrew L. Hook1, Chien-Yi Chang2, Jing Yang1, David J. Scurr1, Robert Langer3, Daniel G. Anderson3, Steve Atkinson2, Paul Williams2, Martyn C. Davies1, Morgan R. Alexander1 1Laboratory of Biophysics and Surface Analysis, University of Nottingham, 2School of Molecular Medical Sciences, University of Nottingham, 3David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.