University of Copenhagen View Institution's Website 54 articles published in JoVE Biology A Thermoplasmonic Approach for Investigating Plasma Membrane Repair in Living Cells and Model Membranes Helena Maria D. Danielsen1, Mohammad Reza Arastoo1, Guillermo Moreno-Pescador1,2, Poul Martin Bendix1 1The Niels Bohr Institute, University of Copenhagen, 2Department of Plant and Environmental Sciences, University of Copenhagen The thermoplasmonic puncture method integrates confocal microscopy, optical tweezers, and gold nanoparticles to study protein responses during plasma membrane repair in cells and giant unilamellar vesicles. The technique enables rapid and localized membrane puncture, allowing the identification of key proteins and their functional roles in the intricate plasma membrane repair machinery. Medicine Doppler Ultrasound-Based Leg Blood Flow Assessment During Single-Leg Knee-Extensor Exercise in an Uncontrolled Setting Jacob Peter Hartmann1,2,3, Rikke Krabek1, Stine B. Nymand1,3, Helene Hartmeyer1, Lasse Gliemann4, Ronan M. G. Berg1,2,3,5, Ulrik Winning Iepsen1,6 1Centre for Physical Activity Research, Copenhagen University Hospital, 2Department of Clinical Physiology and Nuclear Medicine, Copenhagen University Hospital, 3Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 4Department of Nutrition, Exercise and Sports, Integrated Physiology Group, 5Neurovascular Research Laboratory, Faculty of Life Sciences and Education, University of South Wales, 6Department of Anaesthesiology and Intensive Care, Copenhagen University Hospital, Hvidovre Hospital This test-retest study evaluated leg blood flow measured by the Doppler ultrasound technique during single-leg knee-extensor exercise. The within-day, between-day, and inter-rater reliability of the method was investigated. The approach demonstrated high within-day and acceptable between-day reliability. However, the inter-rater reliability was unacceptably low during rest and at low workloads. Genetics Mapping Mammalian 3D Genome Interactions with Micro-C-XL Mariia Metelova1, Rikke Rejnholdt Jensen1, Nils Krietenstein1 1Novo Nordisk Foundation Center for Protein Research, University of Copenhagen A protocol for mapping the three-dimensional genome organization with nucleosome resolution using the genome-wide chromosome conformation capture method Micro-C-XL is presented here. Medicine Hepatic Glucose Production, Ureagenesis, and Lipolysis Quantified using the Perfused Mouse Liver Model Marie Winther-Sørensen1,2,3, Ida Marie Kemp1,3, Hanne Cathrine Bisgaard4, Jens Juul Holst1,5, Nicolai J. Wewer Albrechtsen2,3 1Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 2NNF Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 3Department for Clinical Biochemistry, Copenhagen University Hospital - Bispebjerg and Frederiksberg, 4Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, 5NNF Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen Here, we present a robust method for in situ perfusion of the mouse liver to study the acute and direct regulation of liver metabolism without disturbing the hepatic architecture but in the absence of extra-hepatic factors. Bioengineering Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs Janko Kajtez1, Carmen Radeke2, Johan Ulrik Lind2, Jenny Emnéus3 1Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), University of Copenhagen, 2Department of Health Technology (DTU Health Tech), Technical University of Denmark, 3Department of Biotechnology and Biomedicine (DTU Bioengineering), Technical University of Denmark This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity. Biochemistry Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy Rasmus Jensen1, Niels Ørtenblad2, Cristiano di Benedetto3, Klaus Qvortrup3, Joachim Nielsen2 1Research 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 Straten1,3, Claus Desler2, Marlies J. W. Peeters1 1National 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 Illa1,2, Sarah Baumgarten1, Dennis Danielsen1, Karin Larsen3, Torben Elm1, Peter B. Johansen4, Tom Knudsen5, Brian Lauritzen1, Mikael Tranholm6, Carsten D. Ley1 1Global 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 Kaltenpoth1,2, Laura V. Flórez1,3 1Department 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. Biology Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion Jing Zhang*1, Jing Huang*2, Ishani Majumdar1, Ryan C. James3, Julia Gichimu1, Manikandan Paramasivam4, Durga Pokharel5, Himabindu Gali6, Marina A. Bellani1, Michael M Seidman1 1Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 2Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, 3Department of Molecular Biology and Genetics, Cornell University, 4Department of Cellular and Molecular Medicine, University of Copenhagen, 5Horizon Discovery, 6Boston University School of Medicine While replication fork collisions with DNA adducts can induce double strand breaks, less is known about the interaction between replisomes and blocking lesions. We have employed the proximity ligation assay to visualize these encounters and to characterize the consequences for replisome composition. Neuroscience Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model Jesper Peter Bömers1,2, Sara Ellinor Johansson2, Lars Edvinsson2,4, Tiit Illimar Mathiesen1,3,5, Kristian Agmund Haanes2 1Department of Neurosurgery, Rigshospitalet, 2Department of Clinical Experimental Research, Glostrup Research Institute, Rigshospitalet, 3Department of Clinical Medicine, University of Copenhagen, 4Department of Clinical Sciences, Division of Experimental Vascular Research, Lund University, 5Department of Clinical Neuroscience, Karolinska Institutet Subarachnoid hemorrhage continues to carry a high burden of mortality and morbidity in man. To facilitate further research into the condition and its pathophysiology, a pre-chiasmatic, single injection model is presented. Cancer Research Decellularization of the Murine Cardiopulmonary Complex Alejandro E. Mayorca-Guiliani1, Maria Rafaeva1, Oliver Willacy1, Chris D. Madsen2, Raphael Reuten1, Janine T. Erler1 1Biotech 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. Wojtaszewski1 1Section 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 Zhang1 1Department 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 Anabire1,2,3, Lars Hviid1,4 1Centre 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 Brodin1 1Department 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 Siryaporn1,2, Nina Molin Høyland-Kroghsbo3 1Department 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 Kühl1,4, Klaus Koren5 1Marine 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-Jensen1 1Nordic 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 Zhang1,2, Benjamin S. Rayner1,2, Mathias Jensen3, Clare L. Hawkins1,2,3 1Heart 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 Nedergaard1,2 1Center 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. Pedersen1 1Section 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 Lauritzen1,2 1Department 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 Kissow1,2 1Department 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 Herth1,2,3, Petrine Wellendorph1 1Department 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 Rehfeld1,2, Dorte Louise Egeberg Palme1,2, Kristian Almstrup1,2, Anders Juul1,2, Niels Erik Skakkebaek1,2 1Department 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. Holst1 1Department 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. Jeuken1 1School 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. Innocent1 1Centre 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. 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. Immunology and Infection Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP) Jordan F. Hastings1, Jeremy Z.R. Han1, Robert F. Shearer1,2, Sean P. Kennedy1,3, Mary Iconomou1,4, Darren N. Saunders5, David R. Croucher1,6,7 1The 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 Sanggaard1,2, Nanhong Lou3, Iben Lundgaard3,4, Maiken Nedergaard1,3 1Center 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 Barrès1 1The 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 Arenz1,2 1Nano-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 Arnold1 1School 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. Fang1,6, Konstantinos Palikaras2, Nuo Sun3, Elayne M. Fivenson1, Ryan D. Spangler4, Jesse S. Kerr1, Stephanie A. Cordonnier1, Yujun Hou1, Eszter Dombi5, Henok Kassahun6, Nektarios Tavernarakis2,7, Joanna Poulton5, Hilde Nilsen6, Vilhelm A. Bohr1,8 1Laboratory 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 Rickhag1 1Molecular 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-Olesen1 1Department 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 Svenningsen1 1Department 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 Bisgaard1 1Copenhagen 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 Sakuragi3 1Carlsberg 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 Pakkenberg1,2 1Research 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 Leukel1,2, Jens Bo Nielsen3,4, Jesper Lundbye-Jensen3,4 1Department 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 Dale3 1The 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. Willats2 1Department 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 Lecampion1,2,3, Maïna Floris1,2,3,4, Jean Raphaël Fantino5,6, Christophe Robaglia1,2,3, Christophe Laloi1,2,3 1Laboratoire 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 Regenberg1 1Department 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 Lykkesfeldt1 1Department 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-Botton1,2 1Ecole 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 Aune1,2,3, Lauren Ruiz1, Shingo Kajimura1 1UCSF 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. Moller1,2, Filomena A. Pettolino3, Charlie Hart1, Edwin R. Lampugnani1, William G.T. Willats4, Antony Bacic1,2 1Australian 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 Ronander1,2, Dominique C. Bengtsson1,2, Louise Joergensen1,2, Anja T. R. Jensen1,2, David E. Arnot1,2,3 1Centre 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. Vaegter1 1The 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 Regenberg2 1Department 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).
