University of Munster 14 articles published in JoVE Cancer Research A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies Ana C. Martins Cavaco1,2, Johannes A. Eble1 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. 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 Kudruk1,3, Nicole Heitzig1,3, Hans-Joachim Galla2,3, Volker Gerke1,3, Ursula Rescher1,3 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. Biology Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells Timo Appelhans1, Felix R.M. Beinlich1, Christian P. Richter1, Rainer Kurre2, Karin B. Busch1,3 1School of Biology, University of Osnabrück, 2Center of Cellular Nanoanalytics, Integrated Bioimaging Facility, University of Osnabrück, 3Department of Biology, WWU Münster Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm. Biochemistry Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction Johannes A. Eble1 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. Environment Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS Zhiyong Lin1,3, Xiaoming Sun1,2,3,4, Jörn Peckmann5, Yang Lu2,3, Harald Strauss6, Li Xu2,3, Hongfeng Lu7, Barbara M.A. Teichert6 1School of Earth Sciences and Engineering, Sun Yat-sen University, 2School of Marine Sciences, Sun Yat-sen University, 3Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, 4South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, 5Institut für Geologie, Universität Hamburg, 6Institut für Geologie und Paläontologie, Westfälische Wilhelms-Universität Münster, 7Guangzhou Marine Geological Survey Analyses of the sulfur isotopic composition (δ34S) of pyrite from methane-bearing sediments have typically focused on bulk samples. Here, we applied secondary ion mass spectroscopy to analyze the δ34S values of various pyrite generations to understand the diagenetic history of pyritization. Biology A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions Tanumoy Saha1, Isabel Rathmann1, Milos Galic1 1DFG Cluster of Excellence 'Cells in Motion', (EXC 1003), Institute of Medical Physics and Biophysics, University of Muenster We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions. Immunology and Infection An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction Ivan Kuzmanov1, Alexander M. Herrmann1, Hans-Joachim Galla2, Sven G. Meuth1, 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. Medicine Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing Philipp Lenz1,2, Markus Brückner1, Steffi Ketelhut3, Jan Heidemann4, 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. Medicine A Step by Step Protocol for Subretinal Surgery in Rabbits Sami Al-Nawaiseh1, Fabian Thieltges1, Zengping Liu1,2, Claudine Strack1, Ralf Brinken1, Norbert Braun3, Marc Wolschendorf3, Arvydas Maminishkis5, Nicole Eter4, Boris V. Stanzel1,6 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. Immunology and Infection An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices Kerstin Göbel1, Stefan Bittner1,2, Manuela Cerina3, Alexander M. Herrmann1, Heinz Wiendl1, Sven G. Meuth1,3 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. Neuroscience FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis Benjamin Risse*1,2, Nils Otto*1, Dimitri Berh1,2, Xiaoyi Jiang2, Christian Klämbt1 1Institute of Neuro and Behavioral Biology, Westfälische Wilhelms-Universität Münster, 2Department of Mathematics and Computer Science, Westfälische Wilhelms-Universität Münster FIM is a novel, cost effective imaging system designed to track small moving objects such as C. elegans, planaria or Drosophila larvae. The accompanying FIMTrack program is designed to deliver fast and efficient data analysis. Together, these tools allow high-throughput analysis of behavioral traits. Neuroscience Isolation of Primary Murine Brain Microvascular Endothelial Cells Tobias Ruck1, Stefan Bittner1,2, Lisa Epping1, Alexander M. Herrmann1, Sven G. Meuth1,3 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. Immunology and Infection Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice Stefan Bittner1,2, Ali M. Afzali1, Heinz Wiendl1, Sven G. Meuth1,3 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. Biology A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry Kerstin Trompelt1, Janina Steinbeck1, Mia Terashima1,2, Michael Hippler1 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.