University of Oslo View Institution's Website 18 articles published in JoVE Biology Rapid, Cost-Efficient, Enzyme-Free Passaging of Human Pluripotent Stem Cells on Feeder Cells by Ethylenediaminetetraacetic Acid-Mediated Dis-Adhesion Hege Brincker Fjerdingstad1, Joel C. Glover1,2 1Norwegian Center for Stem Cell Research, Department of Immunology and Transfusion Medicine, Oslo University Hospital, 2Laboratory of Neural Development and Optical Recording (NDEVOR), Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo To avoid the limitations associated with the enzymatic or mechanical passaging of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) cultured on feeder cells, we have established a fast, effective, cost-efficient, high-yield method for harvesting hESC or hiPSC colonies maintained on a feeder cell layer of human foreskin fibroblasts using EDTA-mediated dis-adhesion. Chemistry Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins Huan Bao Ngo1, Inger Oulie1, Léon Reubsaet1, Trine Grønhaug Halvorsen1 1Department of Pharmacy, University of Oslo A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry. Medicine A Piglet Perinatal Asphyxia Model to Study Cardiac Injury and Hemodynamics after Cardiac Arrest, Resuscitation, and the Return of Spontaneous Circulation Eydis Oddsdottir Stenersen1,2, Annette Olsen2, Maria Melheim3, Rønnaug Solberg3,4, Ingrid Dannevig5, Georg Schmölzer6,7, Po-Yin Cheung6,7, Britt Nakstad8,9, Ola Didrik Saugstad10,11, Arild Rønnestad1,2, Anne Lee Solevåg2 1Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, 2Department of Neonatal Intensive Care, Division of Pediatric and Adolescent Medicine, Oslo University Hospital Rikshospitalet, 3Department of Pediatric Research, Division of Paediatric and Adolescent Medicine, Oslo University Hospital Rikshospitalet, 4Department of Pediatrics, Vestfold Hospital Trust, 5Department of Anesthesiology, Oslo University Hospital Rikshospitalet, 6Department of Pediatrics, Faculty of Medicine and Dentistry, University of Alberta, 7Centre for the Studies of Asphyxia and Resuscitation, Neonatal Research Unit, Royal Alexandra Hospital, 8Division of Paediatric and Adolescent Medicine, Institute of Clinical Medicine, University of Oslo, 9Department of Paediatrics and Adolescent Health, University of Botswana, 10Department of Pediatric Research, Oslo University Hospital, University of Oslo, 11Department of Pediatrics, Robert H Lurie Medical Research Center, Northwestern University Chicago This piglet model involves surgical instrumentation, asphyxiation until the cardiac arrest, resuscitation, and post-resuscitation observation. The model allows for multiple sampling per animal, and by using continuous invasive arterial blood pressure, ECG, and non-invasive cardiac output monitoring, it provides knowledge about hemodynamics and cardiac pathophysiology in perinatal asphyxia and neonatal cardiopulmonary resuscitation. Bioengineering Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care Lyubomyr Lytvynchuk*1,2, Zbynek Stranak*3, Hana Studenovska4, David Rais4, Štěpán Popelka4, Lucie Tichotová5,6, Yaroslav Nemesh5,6, Anastasiia Kolesnikova5,6, Ruslan Nyshchuk5,6, Anna Brymová5,6, Zdeňka Ellederová5, Jana Čížková5, Jana Juhásová5, Štefan Juhás5, Pavla Jendelová7, Richárd Nagymihály8, Igor Kozak9, Slaven Erceg10, Susanne Binder11, Brigitte Müller1, Knut Stieger1, Jan Motlik5, Goran Petrovski*8, Taras Ardan*5 1Eye Clinic, Department of Ophthalmology, University Hospital Giessen and Marburg GmbH, 2Karl Landsteiner Institute for Retinal Research and Imaging, 3Department of Ophthalmology, University Hospital Kralovske Vinohrady and Third Faculty of Medicine, Charles University in Prague, 4Institute of Macromolecular Chemistry, Czech Academy of Sciences, 5Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 6Department of Cell Biology, Faculty of Science, Charles University, 7Institute of Experimental Medicine, Czech Academy of Sciences, 8Center for Eye Research, Department of Ophthalmology, Oslo University Hospital and Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, 9Moorfields Eye Hospitals UAE, 10Stem Cell Therapies in Neurodegenerative Diseases Lab, Research Center “Principe Felipe”, 11Eye Center Donaustadt, Department of Ophthalmology, Sigmund Freud University The subretinal implantation of retinal pigmented epithelium (RPE) is one of the most promising approaches for the treatment of degenerative retinal diseases. However, the performance of preclinical studies on large-eye animal models remains challenging. This report presents guidelines for the subretinal transplantation of RPE on a cell carrier into minipigs. Chemistry Analyzing Melts and Fluids from Ab Initio Molecular Dynamics Simulations with the UMD Package Razvan Caracas1,2, Anais Kobsch1, Natalia V. Solomatova1, Zhi Li1, Francois Soubiran1,3, Jean-Alexis Hernandez1,2 1Ecole Normale Supérieure de Lyon, Laboratory of Geology of Lyon UMR5276, CNRS, 2Centre for Earth Evolution and Dynamics (CEED), University of Oslo, 3 Melts and fluids are ubiquitous vectors of mass transport in natural systems. We have developed an open-source package to analyze ab initio molecular-dynamics simulations of such systems. We compute structural (bonding, clusterization, chemical speciation), transport (diffusion, viscosity) and thermodynamic properties (vibrational spectrum). Behavior Using Facial Electromyography to Assess Facial Muscle Reactions to Experienced and Observed Affective Touch in Humans Anbjørn Ree1, India Morrison2, Håkan Olausson2, Uta Sailer1, Markus Heilig2, Leah M. Mayo2 1Department of Behavioral Sciences in Medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, 2Center for Social and Affective Neuroscience, Department of Clinical and Experimental Medicine, Linköping University We describe a protocol to assess facial muscle activity in response to experienced and observed tactile stimulation using facial electromyography. Bioengineering Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum Elif Senem Köksal1, Patrícia F. Belletati1, Ganna Reint1, Ragni Olsson1, Kira D. Leitl1, Ilayda Kantarci1, Irep Gözen1,2,3 1Centre for Molecular Medicine Norway, Faculty of Medicine, University of Oslo, 2Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Oslo, 3Department of Chemistry and Chemical Engineering, Chalmers University of Technology Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum. Cancer Research Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery Sigrid S. Skånland1,2 1Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo, 2K. G. Jebsen Centre for B cell malignancies and K. G. Jebsen Centre for Cancer Immunotherapy, University of Oslo Here, a protocol for medium- to high-throughput analysis of protein phosphorylation events at the cellular level is presented. Phospho flow cytometry is a powerful approach to characterize signaling aberrations, identify and validate biomarkers, and assess pharmacodynamics. Genetics Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes Athanasios Saragliadis1, Thomas Trunk1, Jack C. Leo1 1Evolution and Genetics, Department of Biosciences, University of Oslo Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion constructs as a basis for making deletion mutants in other E. coli strains. Such deletion mutations can be mobilized and inserted into the corresponding locus of a recipient strain using P1 bacteriophage transduction. Neuroscience Preparation of a High-quality Primary Cell Culture from Fish Pituitaries Eirill Ager-Wick1, Kjetil Hodne1, Romain Fontaine1, Kristine von Krogh1, Trude M. Haug2, Finn-Arne Weltzien1 1Department of Basic Sciences and Aquatic Medicine, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, 2Department of Oral Biology, Faculty of Dentistry, University of Oslo Here we describe a protocol to prepare and maintain primary pituitary cell cultures from medaka (Oryzias latipes). The optimized conditions in this protocol take important parameters such as temperature, osmolality, and pH into consideration by mimicking the physiological conditions of the fish, thereby enabling physiologically more meaningful results. Biology The Lactate Dehydrogenase Sequestration Assay — A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells Morten Luhr*1, Paula Szalai*1, Nikolai Engedal1 1The Autophagy Team, Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels. Neuroscience Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy Lauritz Hagen Kennedy1, Johanne Egge Rinholm1,2 1Division of Anatomy, Institute of Basic Medical Sciences, University of Oslo, 2Department of Microbiology, Oslo University Hospital Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented. Medicine The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo Ane M. Holme1,2, Maia B. Holm1,2, Marie C. P. Roland1,3, Hildegunn Horne1,2, Trond M. Michelsen1,3, Guttorm Haugen2,4, Tore Henriksen1,2 1Department of Obstetrics, Oslo University Hospital, 2Institute of Clinical Medicine, University of Oslo, 3Norwegian Advisory Unit on Women's Health, Oslo University Hospital, 4Department of Fetal Medicine, Oslo University Hospital We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling. Immunology and Infection Flow Cytometry-based Assay for the Monitoring of NK Cell Functions Sara Tognarelli*1,2, Benedikt Jacobs*3,4, Nina Staiger1,2, Evelyn Ullrich1,2 1Childrens Hospital, Department of Pediatric Stem Cell Transplantation and Immunology, Johann Wolfgang Goethe-University, 2LOEWE Center for Cell and Gene Therapy, Johann Wolfgang Goethe-University, 3Institute for Cancer Research, Department of Cancer Immunology, Oslo University Hospital, Radiumhospital, 4The KG Jebsen Center for Cancer Immunotherapy, Institute of Clinical Medicine, University of Oslo A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets. Medicine A Neonatal Mouse Spinal Cord Compression Injury Model Mark Züchner1,2, Joel C. Glover2,3, Jean-Luc Boulland2,3 1Department of Neurosurgery, Oslo University Hospital, 2Norwegian Center for Stem Cell Research, Oslo University Hospital, 3Laboratory of Neural Development and Optical Recording (NDEVOR), Department of Physiology, Institute of Basic Medical Sciences, University of Oslo This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery. Biology Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam Stefano Rubino1,4, Petter Melin3, Paul Spellward2, Klaus Leifer1 1Department of Engineering Sciences, Uppsala University, 2Gatan Inc., 3Department of Microbiology, Swedish University of Agricultural Sciences, 4Physics Department, University of Oslo Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM. Medicine Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer Elizabeth S. Nakasone1,2, Hanne A. Askautrud2,3, Mikala Egeblad2 1Watson School of Biological Sciences, 2Cold Spring Harbor Laboratory, 3Departments of Medical Genetics, University of Oslo and Oslo University Hospital We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution. Biology Xenotransplantation of Human Stem Cells into the Chicken Embryo Jean-Luc Boulland1, Gabor Halasi1, Nedim Kasumacic1, Joel C. Glover1,2 1Department of Physiology, University of Oslo, 2Norwegian Center for Stem Cell Research, University of Oslo In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.
