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University of Münster

18 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach
Saleta Sierra 1, Rolf Kaiser 1, Nadine Lübke 1, Alexander Thielen 2, Eugen Schuelter 1, Eva Heger 1, Martin Däumer 3, Stefan Reuter 4, Stefan Esser 5, Gerd Fätkenheuer 6, Herbert Pfister 1, Mark Oette 7, Thomas Lengauer 2
1Institute of Virology, University of Cologne, 2Max Planck Institute for Informatics, 3Institute for Immune genetics, 4Department of Gastroenterology, Hepatology and Infectiology, University of Duesseldorf, 5Department of Dermatology, University of Essen, 6Department of Internal Medicine, University of Cologne, 7Augustinerinnen Hospital

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

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Medicine

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice
Michael T. Kuhlmann 1, Simon Cuhlmann 2,3, Irmgard Hoppe 1, Rob Krams 3, Paul C. Evans 2, Gustav J. Strijkers 4, Klaas Nicolay 4, Sven Hermann 1, Michael Schäfers 1
1European Institute for Molecular Imaging, Westfälische Wilhelms-University Münster, 2British Heart Foundation Cardiovascular Sciences Unit, Imperial College London , 3Department of Bioengineering, Imperial College London , 4Biomedical Engineering, Eindhoven University of Technology

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

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JoVE Journal

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET
Alexander Grabner *1, Dominik Kentrup *1, Uta Schnöckel 2, Gert Gabriëls 1, Rita Schröter 1, Hermann Pavenstädt 1, Otmar Schober 2, Eberhard Schlatter 1, Michael Schäfers *3, Stefan Reuter *1
1Department of Internal Medicine D, Experimental Nephrology, University of Münster, 2Department of Nuclear Medicine, University of Münster, 3European Institute for Molecular Imaging, University of Münster

We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.

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Biology

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format
Miao Zhang 1, Hans R. Schöler 1,2, Boris Greber 1
1Max Planck Institute for Molecular Biomedicine, 2Medical Faculty, University of Münster

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

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Biology

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry
Kerstin Trompelt 1, Janina Steinbeck 1, Mia Terashima 1,2, Michael Hippler 1
1Institute of Plant Biology and Biotechnology, University of Münster, 2Department of Plant Biology, Carnegie Institution for Science

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

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Immunology and Infection

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice
Stefan Bittner 1,2, Ali M. Afzali 1, Heinz Wiendl 1, Sven G. Meuth 1,3
1Department of Neurology, University of Münster, 2Interdisciplinary Center for Clinical Research (IZKF), Münster, 3Institute of Physiology – Neuropathophysiology, University of Münster

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

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Neuroscience

Isolation of Primary Murine Brain Microvascular Endothelial Cells
Tobias Ruck 1, Stefan Bittner 1,2, Lisa Epping 1, Alexander M. Herrmann 1, Sven G. Meuth 1,3
1Department of Neurology, University of Münster, 2Interdisciplinary Center for Clinical Research (IZKF) Münster, 3Institute of Physiology I — Neuropathophysiology I, University of Münster

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

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Immunology and Infection

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices
Kerstin Göbel 1, Stefan Bittner 1,2, Manuela Cerina 3, Alexander M. Herrmann 1, Heinz Wiendl 1, Sven G. Meuth 1,3
1Department of Neurology, University of Münster, 2Germany and Interdisciplinary Center for Clinical Research (IZKF) Münster, 3Institute of Physiology I - Neuropathophysiology I, University of Münster

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

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Medicine

A Step by Step Protocol for Subretinal Surgery in Rabbits
Sami Al-Nawaiseh 1, Fabian Thieltges 1, Zengping Liu 1,2, Claudine Strack 1, Ralf Brinken 1, Norbert Braun 3, Marc Wolschendorf 3, Arvydas Maminishkis 5, Nicole Eter 4, Boris V. Stanzel 1,6
1Department of Ophthalmology, University of Bonn, 2Department of Ophthalmology, National University of Singapore, 3Geuder AG, 4Department of Ophthalmology, University of Münster, 5Section on Epithelial and Retinal Physiology and Disease, National Eye Institute/National Institutes of Health, 6Surgical Retina Department, Singapore National Eye Centre

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

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Medicine

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing
Philipp Lenz 1,2, Markus Brückner 1, Steffi Ketelhut 3, Jan Heidemann 4, Björn Kemper *3, Dominik Bettenworth *1
1Department of Medicine B, University Hospital Münster, 2Institute of Palliative Care, University Hospital Münster, 3Biomedical Technology Center, University of Münster, 4Department of Gastroenterology, Klinikum Bielefeld

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

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Immunology and Infection

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction
Ivan Kuzmanov 1, Alexander M. Herrmann 1, Hans-Joachim Galla 2, Sven G. Meuth 1, Heinz Wiendl *1, Luisa Klotz *1
1Department of Neurology, University Hospital Münster, 2Institute of Biochemistry, University of Münster

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

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Immunology and Infection

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice
Brian Czaya *1,2, Saurav Singh *1,2, Christopher Yanucil 1,2, Karla Schramm 1,2, Christian Faul 1,2, Alexander Grabner 1
1Katz Family Drug Discovery Center and Division of Nephrology and Hypertension, Department of Medicine, University of Miami Leonard M. Miller School of Medicine, 2Department of Cell Biology and Anatomy, University of Miami Leonard M. Miller School of Medicine

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

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Immunology and Infection

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier
Andreas Schulte-Mecklenbeck 1, Urvashi Bhatia 1, Tilman Schneider-Hohendorf 1, Nicholas Schwab 1, Heinz Wiendl *1, Catharina C. Gross *1
1Department of Neurology with Institute of Translational Neurology, University Hospital Münster

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

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Biochemistry

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction
Johannes A. Eble 1
1Institute of Physiological Chemistry and Pathobiochemistry, University of Münster

A detailed protocol to perform a titration ELISA is described. Moreover, a novel algorithm is presented to evaluate titration ELISAs and to obtain a dissociation constant of binding of a soluble ligand to a microtiter plate-immobilized receptor.

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Biochemistry

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator
Anna Livia L. Matos *1,3, David Grill *1,3, Sergej Kudruk 1,3, Nicole Heitzig 1,3, Hans-Joachim Galla 2,3, Volker Gerke 1,3, Ursula Rescher 1,3
1Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation, University of Münster, 2Institute of Biochemistry, University of Münster, 3Cluster of Excellence 'Cells in Motion', University of Münster

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

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Neuroscience

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells
Thomas Müntefering *1, Alexander P.E. Michels *1, Steffen Pfeuffer 1, Sven G. Meuth *1, Tobias Ruck *1
1Institute for Translational Neurology and Neurology Clinic, University of Muenster

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the "myovascular unit".

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Cancer Research

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies
Ana C. Martins Cavaco 1,2, Johannes A. Eble 1
1Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, 2Instituto de Medicina Molecular, Lisbon

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

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Bioengineering

Dynamic Light-Induced Protein Patterns at Model Membranes
Daniele Di Iorio 1, Seraphine V. Wegner 1
1Institute of Physiological Chemistry and Pathobiochemistry, University of Münster

Here, a protocol is described for generating light-regulated and reversible protein patterns with high spatiotemporal precision at artificial lipid membranes. The method consists of the localized photoactivation of the protein iLID (improved light-inducible dimer) immobilized on model membranes that, under blue light, binds to its partner protein Nano (wild-type SspB).

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