University of Copenhagen

47 articles published in JoVE

• Biochemistry

  
  Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy Rasmus Jensen¹, Niels Ørtenblad², Cristiano di Benedetto³, Klaus Qvortrup³, Joachim Nielsen^{2 1}Research center for applied health science, University College South Denmark, ²Department of Sports Science and Clinical Biomechanics, University of Southern Denmark, ³Department of Biomedical Sciences, Core Facility for Integrated Microscopy, University of Copenhagen A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

• Immunology and Infection

  
  Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells Kasper Mølgaard*¹, Anne Rahbech*¹, Özcan Met¹, Inge Marie Svane¹, Per thor Straten^1,3, Claus Desler², Marlies J. W. Peeters^{1 1}National Center for Cancer Immune Therapy, Department of Oncology, University Hospital Herlev, ²Department of Cellular and Molecular Medicine, Center for Healthy Aging, University of Copenhagen, ³Department of Immunology and Microbiology, Inflammation and Cancer Group, University of Copenhagen Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.

• Biology

  
  Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice Ariadna Carol Illa^1,2, Sarah Baumgarten¹, Dennis Danielsen¹, Karin Larsen³, Torben Elm¹, Peter B. Johansen⁴, Tom Knudsen⁵, Brian Lauritzen¹, Mikael Tranholm⁶, Carsten D. Ley^{1 1}Global Drug Discovery, Novo Nordisk A/S, ²Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, ³Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, ⁴Independent consultant, ⁵Catalyst Biosciences, ⁶Værløse Dyreklinik The refined tail vein transection (TVT) bleeding model in anesthetized mice is a sensitive in vivo method for the assessment of hemophilic bleeding. This optimized TVT bleeding model uses blood loss and bleeding time as endpoints, refining other models and avoiding death as an endpoint.

• Biology

  
  Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles Ramya Ganesan¹, Martin Kaltenpoth^1,2, Laura V. Flórez^{1,3 1}Department of Evolutionary Ecology, Institute of Organismic and Molecular Evolution, Johannes Gutenberg University, ²Department of Insect Symbiosis, Max Planck Institute for Chemical Ecology, ³Department of Plant and Environmental Sciences, Section for Organismal Biology, University of Copenhagen This is an adapted method for identifying candidate insect colonization factors in a Burkholderia beneficial symbiont. The beetle host is infected with a random mutant library generated via transposon mutagenesis, and library complexity after colonization is compared to a control grown in vitro.

• Cancer Research

  
  Decellularization of the Murine Cardiopulmonary Complex Alejandro E. Mayorca-Guiliani¹, Maria Rafaeva¹, Oliver Willacy¹, Chris D. Madsen², Raphael Reuten¹, Janine T. Erler^{1 1}Biotech Research and Innovation Centre (BRIC), University of Copenhagen (UCPH), ²Department of Laboratory Medicine, Division of Translational Cancer Research, Lund University This protocol aims to decellularize the heart and lungs of mice. The resulting extracellular matrix (ECM) scaffolds can be immunostained and imaged to map the location and topology of their components.

• Biology

  
  Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice Rasmus Kjøbsted¹, Kohei Kido¹, Jeppe K. Larsen¹, Nicolas O. Jørgensen¹, Jesper B. Birk¹, Ylva Hellsten², Jørgen F. P. Wojtaszewski^{1 1}Section of Molecular Physiology, Department of Nutrition, Exercise and Sports, University of Copenhagen, ²Section of Integrative Physiology, Department of Nutrition, Exercise and Sports, University of Copenhagen Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

• Biochemistry

  
  Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA) Muriel Leandra Schicketanz¹, Paulina Długosz¹, Yong Everett Zhang^{1 1}Department of Biology, University of Copenhagen The Differential Radial Capillary Action of Ligand Assay (DRaCALA) can be used to identify small ligand binding proteins of an organism by using an ORFeome library.

• Immunology and Infection

  
  Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay Maria del Pilar Quintana¹, Nsoh Godwin Anabire^1,2,3, Lars Hviid^{1,4 1}Centre for Medical Parasitology, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, ²West African Centre for Cell Biology of Infectious Pathogens, Department of Biochemistry, Cell and Molecular Biology, University of Ghana, ³Department of Immunology, Noguchi Memorial Institute for Medical Research, ⁴Centre for Medical Parasitology, Department of Infectious Diseases, Rigshospitalet The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

• Neuroscience

  
  Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies Sofie Hørlyck¹, Hans Christian Cederberg Helms¹, Birger Brodin^{1 1}Department of Pharmacy, The Faculty of Health and Medical Sciences, University of Copenhagen Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

• Biology

  
  Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response Jean-Louis Bru¹, Albert Siryaporn^1,2, Nina Molin Høyland-Kroghsbo^{3 1}Department of Molecular Biology & Biochemistry, University of California, Irvine, ²Department of Physics & Astronomy, University of California, Irvine, ³Department of Veterinary and Animal Sciences, University of Copenhagen We detail a simple method to produce high-resolution time-lapse movies of Pseudomonas aeruginosa swarms that respond to bacteriophage (phage) and antibiotic stress using a flatbed document scanner. This procedure is a fast and simple method for monitoring swarming dynamics and may be adapted to study the motility and growth of other bacterial species.

• Chemistry

  
  Luminescence Lifetime Imaging of O₂ with a Frequency-Domain-Based Camera System Maria Moßhammer*¹, Vincent V. Scholz*², Gerhard Holst³, Michael Kühl^1,4, Klaus Koren^{5 1}Marine Biological Section, Department of Biology, University of Copenhagen, ²Center for Electromicrobiology, Aarhus University, ³PCO AG, ⁴Climate Change Cluster, University of Technology Sydney, ⁵Aarhus University Centre for Water Technology, Section for Microbiology, Department of Bioscience, Aarhus University We describe the use of a novel, frequency-domain luminescence lifetime camera for mapping 2D O2 distributions with optical sensor foils. The camera system and image analysis procedures are described along with the preparation, calibration and application of sensor foils for visualizing the O2 microenvironment in the rhizosphere of aquatic plants.

• Biology

  
  An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants Christian S. Thudium*¹, Amalie Engstrom*^1,2, Solveig S. Groen¹, Morten A. Karsdal¹, Anne-Christine Bay-Jensen^{1 1}Nordic Bioscience, ²Department of Biomedical Sciences, University of Copenhagen Here, we present a protocol describing isolation and culturing of cartilage explants from bovine knees. This method provides an easy and accessible tool to describe tissue changes in response to biological stimuli or novel therapeutics targeting the joint.

• Immunology and Infection

  
  In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages Yunjia Zhang^1,2, Benjamin S. Rayner^1,2, Mathias Jensen³, Clare L. Hawkins^{1,2,3 1}Heart Research Institute, ²Sydney Medical School, University of Sydney, ³Department of Biomedical Sciences, University of Copenhagen Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

• Neuroscience

  
  In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy Amanda M Sweeney*¹, Virginia Plá*¹, Ting Du¹, Guojun Liu¹, Qian Sun¹, Sisi Peng¹, Benjamin A. Plog¹, Benjamin T. Kress¹, Xiaowei Wang¹, Humberto Mestre¹, Maiken Nedergaard^{1,2 1}Center for Translational Neuromedicine, University of Rochester Medical Center, ²Center for Translational Neuromedicine, University of Copenhagen Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

• Cancer Research

  
  Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells Michala G. Rolver¹, Line O. Elingaard-Larsen¹, Stine F. Pedersen^{1 1}Section for Cell Biology and Physiology, Department of Biology, Faculty of Science, University of Copenhagen Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

• Neuroscience

  
  In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette Changsi Cai¹, Stefan A. Zambach¹, Jonas C. Fordsmann¹, Micael Lønstrup¹, Kirsten J. Thomsen¹, Aske G. K. Jensen¹, Martin Lauritzen^{1,2 1}Department of Neuroscience, Faculty of Medicine and Health Science, University of Copenhagen, ²Department of Clinical Neurophysiology, Rigshospitalet We present an optimized local ejection procedure using a glass micro-pipette and a fast two-photon hyperstack imaging method, which allows precise measurement of capillary diameter changes and investigation of its regulation in three dimensions.

• Cancer Research

  
  Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis Anna Billeschou¹, Jenna Hunt¹, Hannelouise Kissow^{1,2 1}Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, ²Novo Nordisk Foundation Center of Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

• Neuroscience

  
  Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets Nane Griem-Krey¹, Anders Bue Klein¹, Matthias Herth^1,2,3, Petrine Wellendorph^{1 1}Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, ²Neurobiology Research Unit and CIMBI, Copenhagen University Hospital, ³Department of Clinical Physiology, Nuclear Medicine & PET, Copenhagen University Hospital The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

• Biology

  
  Medium-throughput Screening Assays for Assessment of Effects on Ca²⁺-Signaling and Acrosome Reaction in Human Sperm Anders Rehfeld^1,2, Dorte Louise Egeberg Palme^1,2, Kristian Almstrup^1,2, Anders Juul^1,2, Niels Erik Skakkebaek^{1,2 1}Department of Growth and Reproduction, Copenhagen University Hospital, ²International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), University of Copenhagen Here, two medium-throughput assays for assessment of effects on Ca²⁺-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca²⁺-signaling and acrosome reaction in human sperm.

• Medicine

  
  Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine Rune E. Kuhre¹, Jens J. Holst^{1 1}Department of Biomedical Sciences and NNF Centre for Basic Metabolic Research, The Panum Institute, University of Copenhagen Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption — the isolated perfused rat small intestine.

• Biochemistry

  
  Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy Ievgen Mazurenko¹, Nikos S. Hatzakis², Lars J.C. Jeuken^{1 1}School of Biomedical Sciences & the Astbury Centre for Structural Molecular Biology, University of Leeds, ²Department of Chemistry and Nano-Science Center, University of Copenhagen Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

• Behavior

  
  A Visual Guide for Studying Behavioral Defenses to Pathogen Attacks in Leaf-Cutting Ants Stephen Nilsson-Møller¹, Michael Poulsen¹, Tabitha M. Innocent^{1 1}Centre for Social Evolution, Section for Ecology and Evolution, Department of Biology, University of Copenhagen We present a visual guide to disease defense behaviors in leaf-cutting ants, with individual clips and accompanying definitions, illustrated in an experimental infection scenario. Our main aim is to help other researchers recognize key defensive behaviors and to provide a common understanding for future research in this field.

• Immunology and Infection

  
  A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo Line L. Holm^1,2,3, Milica Vukmanovic-Stejic⁴, Thomas Blauenfeldt¹, Thomas Benfield^2,3, Peter Andersen¹, Arne N. Akbar⁴, Morten Ruhwald^{1 1}Department 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.

• Immunology and Infection

  
  Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP) Jordan F. Hastings¹, Jeremy Z.R. Han¹, Robert F. Shearer^1,2, Sean P. Kennedy^1,3, Mary Iconomou^1,4, Darren N. Saunders⁵, David R. Croucher^{1,6,7 1}The Kinghorn Cancer Centre, Garvan Institute of Medical Research, ²Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, ³RCSI Molecular Medicine, Royal College of Surgeons in Ireland, ⁴Department of Epigenetics, Max Planck Institute of Immunobiology and Epigenetics, ⁵School of Medical Sciences, University of New South Wales, ⁶St Vincent's Hospital Clinical School, University of New South Wales, ⁷School of Medicine and Medical Science, University College Dublin This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

• Neuroscience

  
  Cannula Implantation into the Cisterna Magna of Rodents Anna L.R. Xavier¹, Natalie Linea Hauglund¹, Stephanie von Holstein-Rathlou¹, Qianliang Li¹, Simon Sanggaard^1,2, Nanhong Lou³, Iben Lundgaard^3,4, Maiken Nedergaard^{1,3 1}Center for Translational Neuromedicine, Division of Glial Therapeutics, University of Copenhagen, ²Department of Anesthesiology, Yale School of Medicine, ³Center for Translational Neuromedicine, Division of Glial Therapeutics, University of Rochester Medical Center, ⁴Department of Experimental Medical Science, Wallenberg Center for Molecular Medicine, Lund University Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

• Genetics

  
  Methodology for Accurate Detection of Mitochondrial DNA Methylation Mie Mechta¹, Lars Roed Ingerslev¹, Romain Barrès^{1 1}The Novo Nordisk Foundation for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

• Environment

  
  On the Preparation and Testing of Fuel Cell Catalysts Using the Thin Film Rotating Disk Electrode Method Masanori Inaba¹, Jonathan Quinson¹, Jan Rudolf Bucher², Matthias Arenz^{1,2 1}Nano-Science Center, Department of Chemistry, University of Copenhagen, ²Department of Chemistry and Biochemistry, University of Bern Preparing and testing Pt/C fuel cell catalysts is subject to continuous discussion in the scientific community with respect to reproducibility and best practice. With the presented work, we intend to present a step-by-step tutorial to make and test Pt/C catalysts, which can serve as benchmark for novel catalyst systems.

• Neuroscience

  
  In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing Brandon M. Wild¹, Renée Morris¹, Mihai Moldovan², Christian Krarup², Arun V. Krishnan³, Ria Arnold^{1 1}School of Medical Science, University of New South Wales, ²Department of Clinical Neurophysiology, Rigshospitalet and the Institute of Neuroscience and Pharmacology, University of Copenhagen, ³Prince of Wales Clinical School, University of New South Wales Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

• Medicine

  
  In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice Evandro F. Fang^1,6, Konstantinos Palikaras², Nuo Sun³, Elayne M. Fivenson¹, Ryan D. Spangler⁴, Jesse S. Kerr¹, Stephanie A. Cordonnier¹, Yujun Hou¹, Eszter Dombi⁵, Henok Kassahun⁶, Nektarios Tavernarakis^2,7, Joanna Poulton⁵, Hilde Nilsen⁶, Vilhelm A. Bohr^{1,8 1}Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, ²Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology - Hellas, ³Center for Molecular Medicine, National Heart Lung and Blood Institute, National Institutes of Health, ⁴Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, ⁵Nuffield Department of Obstetrics and Gynaecology, University of Oxford, ⁶Department of Clinical Molecular Biology, University of Oslo and Akershus University Hospital, ⁷Department of Basic Sciences, Faculty of Medicine, University of Crete, ⁸Danish Center for Healthy Aging, University of Copenhagen Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

• Neuroscience

  
  Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake Kathrine L. Jensen¹, Annika H. Runegaard¹, Pia Weikop², Ulrik Gether¹, Mattias Rickhag^{1 1}Molecular Neuropharmacology and Genetics Laboratory, Lundbeck Foundation Center for Biomembranes in Nanomedicine, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, ²Laboratory of Neuropsychiatry, Psychiatric Center Copenhagen and Department of Neuroscience and Pharmacology, University of Copenhagen Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

• Genetics

  
  Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach Jakob Frimodt-Møller¹, Godefroid Charbon¹, Karen A. Krogfelt², Anders Løbner-Olesen^{1 1}Department of Biology, Section for Functional Genomics and Center for Bacterial Stress Response and Persistence (BASP), University of Copenhagen, ²Department of Microbiology and Infection Control, Statens Serum Institut Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

• Biochemistry

  
  Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli Thomas Søndergaard Stenum¹, Michael A. Sørensen¹, Sine Lo Svenningsen^{1 1}Department of Biology, University of Copenhagen Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

• Immunology and Infection

  
  Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels Helene M. Wolsk¹, Bo L. Chawes¹, Jonathan Thorsen¹, Jakob Stokholm¹, Klaus Bønnelykke¹, Susanne Brix², Hans Bisgaard^{1 1}Copenhagen Prospective Studies on Asthma in Childhood (COPSAC), Herlev and Gentofte Hospital, University of Copenhagen, ²Department of Biotechnology and Biomedicine, Technical University of Denmark This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

• Biochemistry

  
  Determination of the Glycogen Content in Cyanobacteria Alice De Porcellinis¹, Niels-Ulrik Frigaard², Yumiko Sakuragi^{3 1}Carlsberg Research Laboratory, ²Department of Biology, University of Copenhagen, ³Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

• Neuroscience

  
  The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy Mikkel Vestergaard Olesen¹, Esther Kjær Needham¹, Bente Pakkenberg^{1,2 1}Research Laboratory for Stereology and Neuroscience, Bispebjerg-Frederiksberg Hospital, ²Institute of Clinical Medicine, Department of Health and Medical Sciences, University of Copenhagen Here, we present a stereological method, the optical fractionator, used to quantify the formation of new neurons, and their survival, in the rat hippocampus following electroconvulsive stimulation. When correctly implemented, the sensitivity and efficiency of stereological methods ensures accurate estimates with a fixed and predetermined precision.

• Neuroscience

  
  Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans Wolfgang Taube¹, Christian Leukel^1,2, Jens Bo Nielsen^3,4, Jesper Lundbye-Jensen^{3,4 1}Department of Medicine, Movement and Sport Science, University of Fribourg (Switzerland), ²Department of Sport Science, University of Freiburg (Germany), ³Department of Neuroscience and Pharmacology, University of Copenhagen, ⁴Department of Nutrition, Exercise and Sports, University of Copenhagen The aim of the present study was to assess changes in transmission at the corticomotoneuronal synapses in humans after repetitive transcranial magnetic stimulation. For this purpose, an electrophysiological method is introduced that allows assessment of pathway specific corticospinal transmission, i.e. differentiation of fast, direct corticospinal pathways from polysynaptic connections.

• Developmental Biology

  
  Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps Charlotte S.L. Bailey*¹, Robert A. Bone*¹, Philip J. Murray², J. Kim Dale^{3 1}The Danish Stem Cell Center (DanStem), University of Copenhagen, ²Division of Mathematics, University of Dundee, ³Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

• Biochemistry

  
  High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits Julia Schückel*¹, Stjepan Krešimir Kračun*¹, William G. T. Willats^{2 1}Department for Plant and Environmental Sciences, University of Copenhagen, ²School of Agriculture, Food and Rural Development, Newcastle University A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

• Biology

  
  An Easy Method for Plant Polysome Profiling Cécile Lecampion^1,2,3, Maïna Floris^1,2,3,4, Jean Raphaël Fantino^5,6, Christophe Robaglia^1,2,3, Christophe Laloi^{1,2,3 1}Laboratoire de Génétique et Biophysique des Plantes, Aix-Marseille Université, ²UMR 7265 Biologie Végétale & Microbiologie Environnementales, CNRS, ³BIAM, CEA, ⁴Department of Biology, Biocenter, University of Copenhagen, ⁵Laboratoire de Chimie Bactérienne, ⁶CNRS, LCB UMR 7283, Aix Marseille Université This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

• Biology

  
  Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells Henrik D. Møller¹, Rasmus K. Bojsen², Chris Tachibana³, Lance Parsons⁴, David Botstein⁵, Birgitte Regenberg^{1 1}Department of Biology, University of Copenhagen, ²National Veterinary Institute, Technical University of Denmark, ³Group Health Research Institute, ⁴Lewis-Sigler Institute for Integrative Genomics, Princeton University, ⁵Calico Life Sciences LLC This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

• Biology

  
  Non-Terminal Blood Sampling Techniques in Guinea Pigs Malene M. Birck¹, Pernille Tveden-Nyborg¹, Maiken M. Lindblad¹, Jens Lykkesfeldt^{1 1}Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen Though a known model, the guinea pig currently represents a niche in experimental animal sciences and limited data is available on the execution of most procedures. Here we present four different approaches to non-terminal in vivo blood sampling techniques in either conscious or anaesthetized guinea pigs.

• Biology

  
  In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors Chiara Greggio*¹, Filippo De Franceschi*¹, Manuel Figueiredo-Larsen*², Anne Grapin-Botton^{1,2 1}Ecole Polytechnique Fédérale de Lausanne, School of Life Sciences, Swiss Institute for Experimental Cancer Research, ²DanStem, University of Copenhagen The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

• Biology

  
  Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells Ulrike Liisberg Aune^1,2,3, Lauren Ruiz¹, Shingo Kajimura^{1 1}UCSF Diabetes Center and Department of Cell and Tissue Biology, University of California, San Francisco, ²Department of Biology, University of Copenhagen, Denmark, ³National Institute of Nutrition and Seafood Research, Bergen, Norway Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

• Biology

  
  Glycan Profiling of Plant Cell Wall Polymers using Microarrays Isabel E. Moller^1,2, Filomena A. Pettolino³, Charlie Hart¹, Edwin R. Lampugnani¹, William G.T. Willats⁴, Antony Bacic^{1,2 1}Australian Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, ²Plant Cell Biology Research Centre, School of Botany, University of Melbourne, ³CSIRO Plant Industry, Black Mountain Laboratories, ⁴Department of Plant Biology and Biotechnology, University of Copenhagen A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

• Biology

  
  Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH) Elena Ronander^1,2, Dominique C. Bengtsson^1,2, Louise Joergensen^1,2, Anja T. R. Jensen^1,2, David E. Arnot^{1,2,3 1}Centre for Medical Parasitology, Department of International Health, Immunology & Microbiology, Faculty of Health Sciences, University of Copenhagen, ²Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), ³Institute of Infection and Immunology Research, School of Biology, University of Edinburgh Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes ¹. The technique is adaptable and can be used on different types of genes, cells and organisms.

• Medicine

  
  The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice Mette Richner¹, Ole J. Bjerrum², Anders Nykjaer¹, Christian B. Vaegter^{1 1}The Lundbeck Foundation Research Center MIND, Department of Biomedicine, Aarhus University, ²Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.

• Immunology and Infection

  
  Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells Martin Weiss Nielsen¹, Claus Sternberg¹, Søren Molin¹, Birgitte Regenberg^{2 1}Department of Systems Biology, Danish Technical University, ²Department of Biology, University of Copenhagen Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).