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19 ARTICLES PUBLISHED IN JoVE

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Biology

Isolation and Characterization of RNA-Containing Exosomes
Cecilia Lässer *1, Maria Eldh *1, Jan Lötvall 1
1Krefting Research Centre, Department of Internal Medicine, Sahlgrenska Academy, University of Gothenburg

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

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Biology

Rat Mesentery Angiogenesis Assay
Klas C. Norrby 1
1Department of Pathology, Institute of Biomedicine, University of Gothenburg

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

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Immunology and Infection

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat
Fatma Dalgakiran 1, Luci A. Witcomb 1, Alex J. McCarthy 1, George M. H. Birchenough 2, Peter W. Taylor 1
1School of Pharmacy, University College London, 2Mucin Biology Group, University of Gothenburg

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

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Medicine

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status
Wendy E. Heywood *1, Anna Baud *1, Emily Bliss 1, Ernestas Sirka 1, Jonathan M. Schott 2, Henrik Zetterberg 3, Daniela Galimberti 4, Neil J. Sebire 5, Kevin Mills 1
1Centre for Translational Omics, Genetics and Genomic Medicine Deptartment, Great Ormond Street Institute of Child Health, University College London, 2Dementia Research Centre, Institute of Neurology, University College London, 3Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy, University of Gothenburg, 4Neurology Unit, Department of Pathophysiology and Transplantation, University of Milan, 5Great Ormond Street Hospital for Children, University College London

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

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Immunology and Infection

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry
Faezzah Baharom 1, Gregory Rankin 2, Saskia Scholz 1, Jamshid Pourazar 2, Clas Ahlm 3, Anders Blomberg 2, Anna Smed-Sörensen 1
1Immunology and Allergy Unit, Department of Medicine Solna, Karolinska Institutet, 2Division of Medicine, Department of Public Health and Clinical Medicine, Umeå University, 3Divison of Infectious Diseases, Department of Clinical Microbiology, Umeå University

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

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Medicine

Absolute Quantification of Aβ1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure
Josef Pannee 1,2, Kaj Blennow 1,2, Henrik Zetterberg 1,2,3, Erik Portelius 1,2
1Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy at University of Gothenburg, Sahlgrenska University Hospital, 2Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, 3UCL Institute of Neurology, Queen Square

A reference measurement procedure for the absolute quantification of Aβ1-42 in human CSF based on solid-phase extraction and liquid chromatography tandem mass spectrometry is described.

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Neuroscience

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid
Karl T. Hansson 1, Tobias Skillbäck 1,2, Elin Pernevik 1, Jessica Holmén-Larsson 1, Gunnar Brinkmalm 1, Kaj Blennow 1,2, Henrik Zetterberg 1,2,3, Johan Gobom 1,2
1Inst. of Neuroscience and Physiology, Dept. of Psychiatry and Neurochemistry, Sahlgrenska Academy at University of Gothenburg, 2Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, 3Department of Molecular Neuroscience, UCL Institute of Neurology

A method for mass spectrometric analysis of endogenous peptides in human cerebrospinal fluid (CSF) is presented. By employing molecular weight cut-off filtration, chromatographic pre-fractionation, mass spectrometric analysis and a subsequent combination of peptide identification strategies, it was possible to expand the known CSF peptidome nearly ten-fold compared to previous studies.

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Cancer Research

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization
Shukun Chen *1, Amin El-Heliebi *1, Julia Schmid 1, Karl Kashofer 2, Zbigniew T. Czyż 3, Bernhard Michael Polzer 3, Klaus Pantel 4, Thomas Kroneis 1,5, Peter Sedlmayr 1
1Institute of Cell Biology, Histology and Embryology, Medical University of Graz, 2Institute of Pathology, Medical University of Graz, 3Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, 4Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, 5Sahlgrenska Cancer Center, University of Gothenburg

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

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Neuroscience

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis
Hoda Fathali 1, Johan Dunevall 1, Soodabeh Majdi 2, Ann-Sofie Cans 1
1Department of Chemistry and Chemical Engineering, Chalmers University of Technology, 2Department of Chemistry and Molecular Biology, University of Gothenburg

Osmotic stress affects exocytosis and the amount of neurotransmitter released during this process. We demonstrate how combining electrochemical methods together with transmission electron microscopy can be used to study the effect of extracellular osmotic pressure on exocytosis activity, vesicle quantal size, and the amount of neurotransmitter released during exocytosis.

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Biology

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA
Katrin Kreisel 1, Martin K.M. Engqvist 1,2, Anders R. Clausen 1
1Department for Medical Biochemistry and Cell Biology, University of Gothenburg, 2Department of Biology and Biological Engineering, Chalmers University of Technology

Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5´-end sequencing.

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JoVE Core

Kinematic Analysis Using 3D Motion Capture of Drinking Task in People With and Without Upper-extremity Impairments
Margit Alt Murphy 1,3, Steve Murphy 2, Hanna C. Persson 1,3, Ulla-Britt Bergström 3, Katharina Stibrant Sunnerhagen 1
1Institution of Neuroscience and Physiology, Rehabilitation Medicine, Sahlgrenska Academy, University of Gothenburg, 2Securesoft Sweden, 3Departement of Occupational Therapy and Physiotherapy, Sahlgrenska University Hospital

This protocol describes an objective method to evaluate the movement performance and sensorimotor function of the upper extremity applied to individuals with stroke and healthy controls. A standardized test procedure, kinematic analysis and outcome variables for three-dimensional motion capture of drinking task are provided.

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Bioengineering

Decellularization and Recellularization Methodology for Human Saphenous Veins
Vijay Kumar Kuna 1, Bo Xu 1, Suchitra Sumitran-Holgersson 1
1Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg

Here, we describe a protocol for the saphenous vein decellularization using detergents and recellularization by perfusion of the peripheral blood and the endothelial medium.

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Environment

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression
Per R. Jonsson 1, Anna-Lisa Wrange 3, Ulrika Lind 2, Anna Abramova 2, Martin Ogemark 1, Anders Blomberg 2
1Department of Marine Sciences, University of Gothenburg, 2Department of Chemistry and Molecular Biology, University of Gothenburg, 3IVL Swedish Environmental Research Institute

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

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Bioengineering

Isolation and Decellularization of a Whole Porcine Pancreas
Vijay Kumar Kuna *1, Niclas Kvarnström *2, Erik Elebring 1, Suchitra Sumitran Holgersson 1
1Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, 2The Transplant Institute, Sahlgrenska University Hospital

Tissue engineering of the whole pancreas is a challenge because of its exocrine and endocrine functions. We show a method for the dissection of an intact porcine pancreas and the process of successful decellularization by perfusion of detergents Triton X-100, sodium deoxycholate, and deoxyribonuclease.

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Engineering

Safe Experimentation in Optical Levitation of Charged Droplets Using Remote Labs
Daniel Galán 1, Oscar Isaksson 2, Jonas Enger 2, Mats Rostedt 2, Andreas Johansson 2, Dag Hanstorp 2, Luis de la Torre 1
1Departamento de Informática y Automática, UNED, 2Department of Physics, University of Gothenburg

Optical levitation is a method for levitating micrometer-sized dielectric objects using laser light. Utilizing computers and automation systems, an experiment on optical levitation can be controlled remotely. Here, we present a remotely controlled optical levitation system that is used both for educational and research purposes.

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JoVE Core

Spectral and Angle-Resolved Magneto-Optical Characterization of Photonic Nanostructures
Mikko Kataja 1, Rafael Cichelero 1,2, Gervasi Herranz 1
1Institut de Ciència de Materials de Barcelona, 2Department of Physics, University of Gothenburg

Photonic band structure enables understanding how confined electromagnetic modes propagate within a photonic crystal. In photonic crystals that incorporate magnetic elements, such confined and resonant optical modes are accompanied by enhanced and modified magneto-optical activity. We describe a measurement procedure to extract the magneto-optical band structure by Fourier space microscopy.

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Biology

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)
Ida Alexandersson 1, Matthew J. Harms 1, Jeremie Boucher 1,2,3
1Bioscience Metabolism, Research and Early Development, Cardiovascular, Renal and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, 2The Lundberg Laboratory for Diabetes Research, University of Gothenburg, 3Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

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Neuroscience

Event Related Potentials (ERPs) and other EEG Based Methods for Extracting Biomarkers of Brain Dysfunction: Examples from Pediatric Attention Deficit/Hyperactivity Disorder (ADHD)
Geir Ogrim 1,2,3, Juri D. Kropotov 4,5
1Neuropsychiatric Team, Åsebråten Outpatient Clinic, Østfold Hospital Trust, 2Institute of Psychology, Norwegian University of Science and Technology, 3Gillberg Neuropsychiatry Centre, University of Gothenburg, 4P. Bechtereva Institute of the Human Brain, Russian Academy of Sciences, 5Department of Neuropsychology, Andrzej Frycz-Modrzewski Krakow University

EEG-methods are applied for extracting biomarkers of brain dysfunctions. The focus is on multi-channel event-related potentials (ERPs) recorded in a cued GO/NOGO task. Non-brain artifacts are corrected and ERPs are compared with the normative data. Examples relate to biomarkers for ADHD diagnosis and prediction of medication response.

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Neuroscience

A Neonatal Rodent Model of Retroorbital Vein Injection
Eridan Rocha-Ferreira 1, Syam Nair 1, Owen Herrock 1, Erik Axel Andersson 2, Carl Joakim Ek 2, Carina Mallard 2, Henrik Hagberg 1
1Centre of Perinatal Medicine and Health, Institute of Clinical Sciences, Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, 2Centre of Perinatal Medicine and Health, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg

This protocol aims to demonstrate a reproducible venous administration route that can be used in rats and mice throughout the neonatal period. This procedure is important for preclinical rodent studies that wish to mirror drug administration in neonatal care units primarily using intravenous administration.

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