RWTH Aachen University View Institution's Website 32 articles published in JoVE Chemistry Screening of Coatings for an All-Solid-State Battery Using In Situ Transmission Electron Microscopy Shibarata Basak1,2, Junbeom Park1, Janghyun Jo2, Osmane Camara1, Amir H. Tavabi2, Hermann Tempel1, Hans Kungl1, Chandramohan George3, Rafal E. Dunin-Borkowski2, Joachim Mayer2,4, Rüdiger-A. Eichel1,5 1Institute of Energy and Climate Research - Fundamental Electrochemistry (IEK-9), Forschungszentrum Jülich GmbH, 2Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, Forschungszentrum Jülich GmbH, 3Dyson School of Design Engineering, Imperial College London, 4Central Facility for Electron Microscopy (GFE), RWTH Aachen University, 5Institute of Physical Chemistry, RWTH Aachen University Utilizing the volume change of Si nanoparticles during (de)lithiation, the present protocol describes a screening method of potential coatings for all-solid-state batteries using in situ transmission electron microscopy. Bioengineering Interlinked Macroporous 3D Scaffolds from Microgel Rods Dirk Rommel1,2, Sitara Vedaraman1,2, Matthias Mork1,2, Laura De Laporte1,2,3 1DWI - Leibniz Institute for Interactive Materials, 2Institute for Technical and Macromolecular Chemistry, RWTH Aachen University, 3Institute of Applied Medical Engineering, Department of Advanced Materials for Biomedicine, RWTH Aachen University Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture. Biochemistry A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro Armand M. G. Jaminon*1, Nikolas Rapp*1, Asim C. Akbulut1, Robert Dzhanaev2, Chris P. Reutelingsperger1, Willi Jahnen-Dechent2, Leon J. Schurgers1,3 1Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 2Helmholtz Institute for Biomedical Engineering, Biointerface Group, RWTH Aachen University, 3Institute of Experimental Medicine and Systems Biology, RWTH Aachen University Cardiovascular disease is the leading cause of death worldwide. Vascular calcification contributes substantially to the burden of cardiovascular morbidity and mortality. This protocol describes a simple method to quantify vascular smooth muscle cell-mediated calcium precipitation in vitro by fluorescent imaging. Medicine Surfactant Depletion Combined with Injurious Ventilation Results in a Reproducible Model of the Acute Respiratory Distress Syndrome (ARDS) Martin Russ*1, Emilia Boerger*1, Philip von Platen2, Roland C. E. Francis1, Mahdi Taher1, Willehad Boemke1, Burkhard Lachmann1, Steffen Leonhardt2, Philipp A. Pickerodt1 1Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt Universität zu Berlin and Berlin Institute of Health Department of Anesthesiology and Intensive Care Medicine, Campus Charité Mitte and Campus Virchow-Klinikum, 2Chair for Medical Information Technology, RWTH Aachen University A combination of surfactant washout using 0.9% saline (35 mL/kg body weight, 37 °C) and high tidal volume ventilation with low PEEP to cause moderate ventilator induced lung injury (VILI) results in experimental acute respiratory distress syndrome (ARDS). This method provides a model of lung injury with low/limited recruitability to study the effect of various ventilation strategies for extended periods. Medicine Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model Adelina Curaj1, Wu Zhoujun1, Mareike Staudt1, Elisa A. Liehn1,2 1Institute for Molecular Cardiovascular Research, RWTH Aachen University, 2Human Genetic Laboratory, University of Medicine and Pharmacy This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved. Neuroscience Brain Death Induction in Mice Using Intra-Arterial Blood Pressure Monitoring and Ventilation via Tracheostomy Paul V. Ritschl*1,2,3, Lena Hofhansel*2,4, Bernhard Flörchinger5, Rupert Oberhuber2, Robert Öllinger1, Johann Pratschke1, Katja Kotsch6 1Department of Surgery Campus Charité Mitte/Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, 2Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, 3Charité Clinician Scientist Program, Berlin Institute of Health, 4Department of Psychiatry, Psychotherapy and Psychosomatics, RWTH Aachen University, 5Department of Cardiothoracic Surgery, University Medical Center Regensburg, 6Department of General, Visceral and Vascular Surgery, Charité - Universitätsmedizin Berlin We present a murine model of brain death induction in order to evaluate the influence of its pathophysiological effects on organs as well as on consecutive grafts in the context of solid organ transplantation. Biochemistry Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study Matthias Knödler1,3, Clemens Rühl2, Patrick Opdensteinen1,3, Johannes F. Buyel1,3 1Institute for Molecular Biotechnology, RWTH Aachen University, 2Sanofi Deutschland GmbH, 3Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V. In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A. Neuroscience Conducting Hyperscanning Experiments with Functional Near-Infrared Spectroscopy Vanessa Reindl1,2, Kerstin Konrad1,2, Christian Gerloff2,3, Jana A. Kruppa1,2,4, Laura Bell1, Wolfgang Scharke1 1Child Neuropsychology Section, Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, Medical Faculty, RWTH Aachen University, 2JARA-Brain Institute II, Molecular Neuroscience and Neuroimaging, RWTH Aachen & Research Centre Juelich, 3Lehrstuhl II für Mathematik, RWTH Aachen University, 4Translational Brain Research in Psychiatry and Neurology, Department of Child and Adolescent Psychiatry, Psychosomatics, and Psychotherapy, University Hospital Aachen The present protocol describes how to conduct fNIRS hyperscanning experiments and analyze brain-to-brain synchrony. Further, we discuss challenges and possible solutions. Developmental Biology An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues Bartosz Ditkowski1, Tiago R Veloso1, Martyna Bezulska-Ditkowska1,2, Andreas Lubig3, Stefan Jockenhoevel3, Petra Mela3, Ramadan Jashari4, Marc Gewillig1, Bart Meyns5, Marc F Hoylaerts2, Ruth Heying1 1Cardiovascular Developmental Biology, Department of Cardiovascular Sciences, KU Leuven, 2Centre for Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, 3Department of Biohybrid & Medical Textiles, AME - Helmholtz Institute for Biomedical Engineering, RWTH Aachen University, 4European Homograft Bank, Saint Jean Clinique, 5Division of Clinical Cardiac Surgery, Department of Cardiovascular Sciences, KU Leuven We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues. Medicine Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo Tim R. Glowka1, Pascal Paschenda2, Michael Czaplik3, Jörg C. Kalff1, René H. Tolba2 1Department of Surgery, University of Bonn, 2Institute for Laboratory Animal Science & Experimental Surgery, RWTH Aachen University, 3Department of Anesthesiology, RWTH Aachen University Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments. Immunology and Infection Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices Vanessa Neuhaus*1, Olga Danov*1, Sebastian Konzok1, Helena Obernolte1, Susann Dehmel1, Peter Braubach2, Danny Jonigk2, Hans-Gerd Fieguth3, Patrick Zardo4, Gregor Warnecke4, Christian Martin5, Armin Braun1,6, Katherina Sewald1 1Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), German Center for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of REBIRTH Cluster of Excellence, 2Institute for Pathology, Hannover Medical School, German Center for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), 3Division of Thoracic and Vascular Surgery, Klinikum Region Hannover (KRH), 4Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, German Center for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), 5Institute of Pharmacology and Toxicology, RWTH Aachen University, 6Institute for Immunology, Hannover Medical School In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances. Bioengineering Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology Johannes Hemmerich*1,2, Lars Freier*1,2, Wolfgang Wiechert1,2,3, Eric von Lieres1,2, Marco Oldiges1,2,4 1IBG-1: Biotechnology, Forschungszentrum Jülich, 2Research Center Jülich, Bioeconomy Science Center (BioSC), 3Computational Systems Biotechnology (AVT.CSB), RWTH Aachen University, 4Institute for Biotechnology, RWTH Aachen University This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation. Biochemistry A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties Johannes F. Buyel1,2, Hannah M. Gruchow1, Martin Wehner3 1Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V., 2Institute for Molecular Biotechnology, RWTH Aachen University, 3Fraunhofer Institute for Laser Technology ILT, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V. A method was developed to determine the specific heat capacity and thermal conductivity of leaf tissue by non-invasive, contact-free near infrared laser probing, which requires less than 1 min per sample. Medicine Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice Chichi Xie1, Weiwei Wei1, Andrea Schenk3, Lars Ole Schwen3, Sara Zafarnia2, Michael Schwier3, Felix Gremse2, Isabel Jank1, Olaf Dirsch4, Uta Dahmen1 1Department of General, Visceral and Vascular Surgery, Jena University Hospital, 2Experimental Molecular Imaging, RWTH Aachen University, 3Fraunhofer Institute for Medical Image Computing MEVIS, 4Institute of Pathology, Klinikum Chemnitz gGmbH Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using µCT and histological serial sections. Neuroscience In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ Tobias Ackels1,2, Daniela R. Drose1, Marc Spehr1 1Department of Chemosensation, Institute for Biology II, RWTH Aachen University, 2Mill Hill Laboratory, The Francis Crick Institute Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings. Chemistry Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels Otto L. J. Virtanen1, Ashvini Purohit1, Monia Brugnoni1, Dominik Wöll1, Walter Richtering1 1Institute of Physical Chemistry, RWTH Aachen University Non-stirred precipitation polymerization provides a rapid, reproducible prototyping approach to the synthesis of stimuli-sensitive poly(N-isopropylacrylamide) microgels of narrow size distribution. In this protocol synthesis, light scattering characterization and single particle fluorescence tracking of these microgels in a wide-field microscopy setup are demonstrated. Biology Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts Johannes F. Buyel1,2, Jürgen Hubbuch3, Rainer Fischer1,2 1Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V., 2Institute for Molecular Biotechnology, RWTH Aachen University, 3Department of Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT) Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 °C. Biology Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts Johannes F. Buyel1,2 1Institute for Molecular Biology and Applied Ecology IME, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V., 2Institute for Molecular Biotechnology, RWTH Aachen University The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing. Bioengineering Hybrid µCT-FMT imaging and image analysis Felix Gremse*1, Dennis Doleschel*1, Sara Zafarnia1, Anne Babler2, Willi Jahnen-Dechent2, Twan Lammers1,3, Wiltrud Lederle1, Fabian Kiessling1 1Experimental Molecular Imaging, RWTH Aachen University, 2Institute for Biomedical Engineering - Biointerface Laboratory, RWTH Aachen University, 3Utrecht Institute for Pharmaceutical Sciences, Utrecht University We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (µCT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution. Medicine Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice Adelina Curaj1, Sakine Simsekyilmaz1, Mareike Staudt1, Elisa Liehn1 1Institute for Molecular Cardiovascular Research, RWTH Aachen University, Germany A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction). Immunology and Infection Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice Felix Heymann*1, Patricia M. Niemietz*1, Julia Peusquens1, Can Ergen1, Marlene Kohlhepp1, Jana C. Mossanen1, Carlo Schneider1, Michael Vogt2, Rene H. Tolba3, Christian Trautwein1, Christian Martin4, Frank Tacke1 1Department of Medicine III, RWTH University-Hospital Aachen, 2IZKF Aachen Core Facility "Two-Photon Imaging", RWTH University-Hospital Aachen, 3Institute for Laboratory Animal Science & Experimental Surgery, RWTH Aachen University, 4Institute for Pharmacology, RWTH University-Hospital Aachen Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring. Medicine Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis Carmen G. Tag1, Sibille Sauer-Lehnen1, Sabine Weiskirchen1, Erawan Borkham-Kamphorst1, René H. Tolba2, Frank Tacke3, Ralf Weiskirchen1 1Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry, RWTH Aachen University, 2Institute for Laboratory Animal Science and Experimental Surgery, RWTH Aachen University, 3Department of Medicine III, RWTH Aachen University Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days. Neuroscience Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings Guanxiao Qi1, Gabriele Radnikow1, Dirk Feldmeyer1,2 1Institute of Neuroscience and Medicine (INM-2), Research Centre Jülich, 2Department of Psychiatry, Psychotherapy and Psychosomatics, Medical Faculty, JARA, RWTH Aachen University Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis. Biology Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues Gisela Maria Hanz*1, Britta Jung*1, Anna Giesbertz1, Matyas Juhasz1, Elmar Weinhold1 1Institute of Organic Chemistry, Department of Chemistry, RWTH Aachen University DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling. Environment Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants Megan Garvey1, Johannes Klinger*1, Holger Klose*1, Rainer Fischer2, Ulrich Commandeur1 1Institute for Molecular Biotechnology, Bio7, RWTH Aachen University, 2Fraunhofer Institute for Molecular Biology and Applied Ecology Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression. Bioengineering Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study Johannes Felix Buyel1, Rainer Fischer1,2 1Institute for Molecular Biotechnology, RWTH Aachen University, 2Institute for Molecular Biology and Applied Ecology, Fraunhofer Gesellschaft We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants. Medicine A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis Sakine Simsekyilmaz1, Fabian Schreiber2, Stefan Weinandy3, Felix Gremse4, Tolga Taha Sönmez5, Elisa A. Liehn1 1Institute for Molecular Cardiovascular Research, RWTH Aachen University, 2Institute for Textile Technology and Mechanical Engineering, RWTH Aachen University, 3Institute for Applied Medical Engineering, Helmholtz-Institute of RWTH Aachen University, 4Department of Experimental Molecular Imaging, RWTH Aachen University, 5Department of Oral and Maxillofacila Surgery, RWTH Aachen University A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis. Medicine Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction Kazuyuki Nagai1,2, Shintaro Yagi2, Shinji Uemoto2, Rene H. Tolba1 1Institute for Laboratory Animal Science and Experimental Surgery, RWTH-Aachen University, 2Department of Hepato-Biliary-Pancreatic Surgery and Transplantation, Graduate School of Medicine, Kyoto University Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft. Medicine Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats Karina Schleimer1, Jochen Grommes1, Andreas Greiner1, Houman Jalaie1, Johannes Kalder1, Stephan Langer1, Thomas A. Koeppel1, Michael Jacobs1, Maria Kokozidou1 1European Vascular Center Aachen-Maastricht, Department of Vascular Surgery, University Hospital RWTH Aachen Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice. Biology Detection of Histone Modifications in Plant Leaves Michal Jaskiewicz1,2, Christoph Peterhansel3, Uwe Conrath2 1Department of Botany, RWTH Aachen University, 2Department of Plant Physiology, RWTH Aachen University, 3Department of Botany, Leibniz University A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes. Biology Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS) Thomas Lenz1, Peter Poot2, Olivia Gräbner1, Mirko Glinski1, Elmar Weinhold2, Mathias Dreger1, Hubert Köster1 1Department of Biochemistry / Analytics, caprotec bioanalytics GmbH, 2Institute of Organic Chemistry, RWTH Aachen University Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS. Biology Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography Matthew C. Johnson*1, Frederik Rudolph*1,2, Tina M. Dreaden*3, Gengxiang Zhao*1, Bridgette A. Barry3, Ingeborg Schmidt-Krey1,3 1School of Biology, Georgia Institute of Technology, 2Department of Molecular Pharmacology, RWTH Aachen University, 3School of Chemistry and Biochemistry, Georgia Institute of Technology Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 – 90kDa.