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19 ARTICLES PUBLISHED IN JoVE

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Biology

Xenotransplantation of Human Stem Cells into the Chicken Embryo
Jean-Luc Boulland 1, Gabor Halasi 1, Nedim Kasumacic 1, Joel C. Glover 1,2
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.

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Biology

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival
Per Wessman 1, Sebastian Håkansson 1, Klaus Leifer 2, Stefano Rubino 2
1Department of Microbiology, Uppsala Biocenter, Swedish University of Agricultural Sciences, 2Department of Engineering Sciences, Uppsala University

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

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Biology

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam
Stefano Rubino 1,4, Petter Melin 3, Paul Spellward 2, Klaus Leifer 1
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.

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Medicine

A Neonatal Mouse Spinal Cord Compression Injury Model
Mark Züchner 1,2, Joel C. Glover 2,3, Jean-Luc Boulland 2,3
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.

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Immunology and Infection

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions
Sara Tognarelli *1,2, Benedikt Jacobs *3,4, Nina Staiger 1,2, Evelyn Ullrich 1,2
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.

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Medicine

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo
Ane M. Holme 1,2, Maia B. Holm 1,2, Marie C. P. Roland 1,3, Hildegunn Horne 1,2, Trond M. Michelsen 1,3, Guttorm Haugen 2,4, Tore Henriksen 1,2
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.

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Neuroscience

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy
Lauritz Hagen Kennedy 1, Johanne Egge Rinholm 1,2
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.

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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 Engedal 1
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.

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Neuroscience

Preparation of a High-quality Primary Cell Culture from Fish Pituitaries
Eirill Ager-Wick 1, Kjetil Hodne 1, Romain Fontaine 1, Kristine von Krogh 1, Trude M. Haug 2, Finn-Arne Weltzien 1
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.

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Genetics

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes
Athanasios Saragliadis 1, Thomas Trunk 1, Jack C. Leo 1
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.

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JoVE Journal

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery
Sigrid S. Skånland 1,2
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.

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Bioengineering

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum
Elif Senem Köksal 1, Patrícia F. Belletati 1, Ganna Reint 1, Ragni Olsson 1, Kira D. Leitl 1, Ilayda Kantarci 1, Irep Gözen 1,2,3
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.

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JoVE Core

Using Facial Electromyography to Assess Facial Muscle Reactions to Experienced and Observed Affective Touch in Humans
Anbjørn Ree 1, India Morrison 2, Håkan Olausson 2, Uta Sailer 1, Markus Heilig 2, Leah M. Mayo 2
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.

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Neuroscience

Pharmacological Validation of the Prepulse Inhibition of Startle Response in Larval Zebrafish using a Commercial Automated System and Software
Nancy Saana Banono 1,2, Camila V. Esguerra 1,2
1Chemical Neuroscience Group, Centre for Molecular Medicine Norway (NCMM), Faculty of Medicine, University of Oslo, 2Section for Pharmacology and Pharmaceutical Biosciences, Department of Pharmacy, Faculty of Mathematics and Natural Sciences, University of Oslo

Here we describe a protocol that utilizes commercially available automated systems to pharmacologically validate the prepulse inhibition (PPI) assay in larval zebrafish.

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Chemistry

Analyzing Melts and Fluids from Ab Initio Molecular Dynamics Simulations with the UMD Package
Razvan Caracas 1,2, Anais Kobsch 1, Natalia V. Solomatova 1, Zhi Li 1, Francois Soubiran 1,3, Jean-Alexis Hernandez 1,2
1Ecole Normale Supérieure de Lyon, Laboratory of Geology of Lyon UMR5276, CNRS, 2Centre for Earth Evolution and Dynamics (CEED), University of Oslo, 3CEA, DAM, DIF

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).

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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 Studenovska 4, David Rais 4, Štěpán Popelka 4, Lucie Tichotová 5,6, Yaroslav Nemesh 5,6, Anastasiia Kolesnikova 5,6, Ruslan Nyshchuk 5,6, Anna Brymová 5,6, Zdeňka Ellederová 5, Jana Čížková 5, Jana Juhásová 5, Štefan Juhás 5, Pavla Jendelová 7, Richárd Nagymihály 8, Igor Kozak 9, Slaven Erceg 10, Susanne Binder 11, Brigitte Müller 1, Knut Stieger 1, Jan Motlik 5, 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.

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Biology

Rapid, Cost-Efficient, Enzyme-Free Passaging of Human Pluripotent Stem Cells on Feeder Cells by Ethylenediaminetetraacetic Acid-Mediated Dis-Adhesion
Hege Brincker Fjerdingstad 1, Joel C. Glover 1,2
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.

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Chemistry

Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins
Huan Bao Ngo 1, Inger Oulie 1, Léon Reubsaet 1, Trine Grønhaug Halvorsen 1
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.

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Medicine

A Piglet Perinatal Asphyxia Model to Study Cardiac Injury and Hemodynamics after Cardiac Arrest, Resuscitation, and the Return of Spontaneous Circulation
Eydis Oddsdottir Stenersen 1,2, Annette Olsen 2, Maria Melheim 3, Rønnaug Solberg 3,4, Ingrid Dannevig 5, Georg Schmölzer 6,7, Po-Yin Cheung 6,7, Britt Nakstad 8,9, Ola Didrik Saugstad 10,11, Arild Rønnestad 1,2, Anne Lee Solevåg 2
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.

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