University of Southern Denmark 13 articles published in JoVE Biochemistry Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures Stephanie Stransky1, Dejauwne Young1, Karoline Mikkelsen2,3, Annemette Præstegaard Thulesen2, Helle Sedighi Frandsen3, Simone Sidoli1 1Department of Biochemistry, Albert Einstein College of Medicine, 2Department of Biochemistry and Molecular Biology, University of Southern Denmark, 3CelVivo ApS We present a protocol for growing high-reproducible spheroids and their phenotypic characterization using image capture and proteomics. 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. Medicine Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy Sabrina T. Hansen1,2, Charlotte H. Jensen2,3,4, Jens A. Sørensen2,4,5, Søren P. Sheikh2,3,4, Lars Lund1,2,4 1Department of Urology, Odense University Hospital, 2Clinical Institute, University of Southern Denmark, 3Laboratory of Molecular and Cellular Cardiology, Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, 4Center for Vascular Regeneration, Odense University Hospital, 5Research Unit for Plastic Surgery, Odense University Hospital Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP). Medicine Direct Drug Delivery to Kidney via the Renal Artery Ulrika Dahlqvist1, Tajana Tesan Tomic2, Magnus Söderberg3, Jane Stubbe4, Camilla Enggaard4, Anette Ericsson5, Alex-Xianghua Zhou1, Anna Björnson Granqvist1, Lena William-Olsson1 1Bioscience Renal, Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, 2Cell/mol Pharmacology, Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, 3CVRM Safety, Safety, Clinical Pharmacology & Safety Sciences, R&D, 4Department of Cardiovascular and Renal Research, University of Southern Denmark, 5Clinical Renal, Late-stage Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse. Medicine A Revised Method for Inducing Secondary Lymphedema in the Hindlimb of Mice Alexander Wiinholt1,2, Mads G. Jørgensen2, Amar Bučan1,2, Farima Dalaei1,2, Jens A. Sørensen2 1University of Southern Denmark, 2Department of Plastic Surgery, Odense University Hospital This animal model enables researchers to induce statistically significant secondary lymphedema in the hindlimb of mice, lasting at least 8 weeks. The model can be used to study the pathophysiology of lymphedema and to investigate novel treatment options. Immunology and Infection Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes Jeff Granhøj1, Henrik Dimke1,2, Per Svenningsen1 1Department of Molecular Medicine, University of Southern Denmark, 2Department of Nephrology, Odense University Hospital Here we present a method to select for novel variants of the E. coli biotin-protein ligase BirA that biotinylates a specific target peptide. The protocol describes the construction of a plasmid for the bacterial display of the target peptide, generation of a BirA library, selection and characterization of BirA variants. Immunology and Infection Quantification of Atherosclerosis in Mice Monica Centa1, Daniel F.J. Ketelhuth1,2, Stephen Malin1, Anton Gisterå1 1Cardiovascular Medicine Unit, Center for Molecular Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital, 2Department of Cardiovascular and Renal Research, Institute for Molecular Medicine, University of Southern Denmark (SDU) Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery. Bioengineering Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells Emma Pipó-Ollé1, Prasad Walke2, Martine K. Notabi1, Rime B. El-Houri1, Morten Østergaard Andersen1, David Needham2,3,4, Eva C. Arnspang1 1Department of Chemical Engineering, Biotechnology and Environmental Technology, University of Southern Denmark, 2Center for Single Particle Science and Engineering (SPSE), Institute for Molecular Medicine, Health Sciences, University of Southern Denmark, 3Department of Mechanical Engineering and Material Science, Duke University, 4School of Pharmacy, University of Nottingham This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique. Biochemistry Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli Patrick Rosendahl Andreassen1, Jens Sivkær Pettersen1, Mikkel Jørgensen1 1Department of Biochemistry and Molecular Biology, University of Southern Denmark Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest. Immunology and Infection Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples Maiken W. Rosenstierne1, Christopher E. Jensen1, Anders Fomsgaard1,2 1Virus Research & Development Laboratory, Statens Serum Institut, 2Infectious Disease Research Unit, University of Southern Denmark Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals). Medicine Cavernous Nerve Stimulation and Recording of Intracavernous Pressure in a Rat Morten Hox1, Travis Mann-Gow2, Lars Lund3,4, Peter Zvara1,4 1Biomedical Laboratory and the Research Unit of Urology, Department of Clinical Research, University of Southern Denmark, 2Department of Surgery, University of Vermont, 3Research Unit of Urology, Department of Clinical Research, University of Southern Denmark, 4Department of Urology, Odense University Hospital This study describes a simplified surgical procedure and technique for performing cavernous nerve stimulation with the isolation of the nerve-electrode complex using silicone glue and intracavernous pressure measurement. Bioengineering Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy Maria Bloksgaard1, Bjarne Thorsted2, Jonathan R. Brewer2, Jo G. R. De Mey1,3 1Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, 2Department of Biochemistry and Molecular Biology, University of Southern Denmark, 3Department of Cardiac, Thoracic and Vascular Surgery, Odense University Hospital We describe simultaneous mechanical testing and 3D-imaging of the arterial wall of isolated, live human resistance arteries, and Fiji and Ilastik image analyses for the quantification of elastin and collagen spatial organization and volume densities. We discuss the use of these data in mathematical models of arterial wall mechanics. Medicine Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model Travis K. Mann-Gow*1, Troy R. Larson*1, Chrissie T. Wøien2, Thomas M. Andersen2, Karl-Erik Andersson3,4, Peter Zvara1,2 1Department of Surgery, University of Vermont, 2Department of Urology and Biomedical Laboratory, University of Southern Denmark, 3Wake Forest Institute for Regenerative Medicine, Wake Forest University, 4Institute for Clinical Medicine, Aarhus University This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization.
