University of Cologne View Institution's Website 28 articles published in JoVE Biochemistry Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation Trinity Cookis1, Paul Sauer2,3, Simon Poepsel4,5, Bong-Gyoon Han6, Dominik A. Herbst1,2,6, Robert Glaeser6, Eva Nogales1,2,3,6 1Department of Molecular and Cell Biology, University of California, Berkeley, 2California Institute for Quantitative Biology (QB3), University of California, Berkeley, 3Howard Hughes Medical Institute, University of California, Berkeley, 4Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital, University of Cologne, 5Cologne Excellence Cluster for Cellular Stress Responses in Ageing-Associated Diseases (CECAD), University of Cologne, 6Molecular Biophysics and Integrative Bio-Imaging Division, Lawrence Berkeley National Laboratory A step-by-step protocol for fabricating streptavidin affinity grids is provided for use in structural studies of challenging macromolecular samples by cryo-electron microscopy. Biology Terminal H-reflex Measurements in Mice Frederique Wieters*1, Matthias Gruhn*2, Ansgar Büschges2, Gereon R. Fink1,3, Markus Aswendt1 1Faculty of Medicine and University Hospital Cologne, Department of Neurology, University of Cologne, 2Department for Animal Physiology, Institute for Zoology, Biocenter Cologne, University of Cologne, 3Cognitive Neuroscience, Institute of Neuroscience and Medicine (INM-3), Research Center Juelich The clinical evaluation of spasticity based on the Hoffmann reflex (H-reflex) and using electrical stimulation of peripheral nerves is an established method. Here, we provide a protocol for a terminal and direct nerve stimulation for H-reflex quantification in the mouse forepaw. Biology In Vitro Analysis of E3 Ubiquitin Ligase Function Leonie Müller1, Carl Elias Kutzner1,2, Vishnu Balaji1, Thorsten Hoppe1,2 1Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD), University of Cologne, 2Center for Molecular Medicine Cologne, University of Cologne The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture. Medicine Simultaneous Laryngopharyngeal and Conventional Esophageal pH Monitoring Dolores T. Müller1, Justus Toader1, Benjamin Babic1, Wolfgang Schröder1, Jessica M. Leers1, Christiane J. Bruns1, Hans F. Fuchs1 1Department of General, Visceral, Cancer and Transplant Surgery, University of Cologne Laryngopharyngeal pH monitoring has been specifically designed to measure acid exposure above the upper esophageal sphincter and complements diagnostic evaluation in patients that present with mainly extraesophageal reflux symptoms. Patients with suspected laryngopharyngeal reflux (LPR) were evaluated using distal esophageal and laryngopharyngeal pH testing simultaneously. Immunology and Infection Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA Marc Herb1, Alina Farid1, Alexander Gluschko1, Martin Krönke1,2,3,4, Michael Schramm1 1Institute for Medical Microbiology, Immunology and Hygiene, Faculty of Medicine and University Hospital Cologne, University of Cologne, 2Center for Molecular Medicine Cologne, 3Cologne Cluster of Excellence on Cellular Stress Responses in Aging-associated Diseases (CECAD), 4German Center for Infection Research (DZIF) Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids. Medicine Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique Martin Ryll1, Julian Bucher1, Moritz Drefs1, Florian Bösch1, K. Kumaraswami2, Tobias Schiergens1, Hanno Niess1, Markus Schoenberg1, Sven Jacob1, Markus Rentsch1, Markus Guba1, Jens Werner1, Joachim Andrassy1, Michael N. Thomas1,3 1Department of General, Visceral, and Transplant Surgery, Ludwig Maximilian University of Munich, 2Walter-Brendel-Centre of Experimental Medicine, Ludwig Maximilian University of Munich, 3Department of General, Visceral and Cancer Surgery, University Hospital of Cologne, University of Cologne Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation. Neuroscience Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice Andreas Lundt1,2, Julien Soos1, Christina Henseler1, Muhammad Imran Arshaad1, Ralf Müller3, Dan Ehninger4, Jürgen Hescheler5, Agapios Sachinidis5, Karl Broich6, Carola Wormuth1,7, Anna Papazoglou1, Marco Weiergräber1 1Experimental Neuropsychopharmacology, Federal Institute for Drugs and Medical Devices, 2KBRwyle GmbH, 3Cognitive Neurophysiology, Department of Psychiatry and Psychotherapy, Faculty of Medicine, University of Cologne, 4Molecular and Cellular Cognition, German Center for Neurodegenerative Diseases (DZNE), 5Institute of Neurophysiology, Faculty of Medicine, University of Cologne, 6Federal Institute for Drugs and Medical Devices (BfArM), 7Thescon GmbH Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice. Developmental Biology Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence Cally Xiao1,2,3, Frank Nitsche4, Hisham Bazzi2,3 1Graduate Program in Pharmacology and Experimental Therapeutics, University of Cologne, 2Department of Dermatology and Venereology, University Hospital of Cologne, 3Cologne Cluster of Excellence in Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 4Department of General Ecology, Institute for Zoology, Biocenter Cologne, University of Cologne The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF). Medicine Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease Louise Benning1, Samuel Robinson1,2, Marie Follo3, Lukas Andreas Heger1, Daniela Stallmann1, Daniel Duerschmied1, Christoph Bode1, Ingo Ahrens1,4, Marcus Hortmann1 1Department of Cardiology and Angiology I, Heart Center Freiburg University, Faculty of Medicine, University of Freiburg, 2Department of Medicine, Monash University, 3Department of Medicine I, Lighthouse Core Facility, Medical Center, University of Freiburg, 4Department of Cardiology, Augustinerinnen Hospital, Academic Teaching Hospital, University of Cologne Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease. Biology A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri) Joanna Dodzian1,3, Sam Kean1, Jens Seidel1, Dario Riccardo Valenzano1,2 1Max Planck Institute for the Biology of Ageing, 2CECAD, University of Cologne, 3International Institute of Molecular and Cell Biology in Warsaw Laboratory housing of turquoise killifish can be scaled up to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance. Immunology and Infection Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes Katharina Dinger1, Jasmine Mohr1, Christina Vohlen1,2, Dharmesh Hirani1, Eva Hucklenbruch-Rother2, Regina Ensenauer3, Jörg Dötsch2, Miguel A. Alejandre Alcazar1,2 1Translational Experimental Pediatrics, Experimental Pulmonology, Department of Pediatrics and Adolescent Medicine, University of Cologne, 2Department of Pediatrics and Adolescent Medicine, University of Cologne, 3Division of Experimental Pediatrics and Metabolism, University Children's Hospital, Heinrich Heine University Düsseldorf The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice. Immunology and Infection Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages Saray Gutiérrez1,2, Martina Wolke2, Georg Plum2, Nirmal Robinson1,2 1Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, 2Cologne Cluster of Excellence in Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin. Medicine Transcanalicular Diode Laser-assisted Dacryocystorhinostomy for the Treatment of Primary Acquired Nasolacrimal Duct Obstruction Joel M. Mor1, Yongwei Guo1, Konrad R. Koch1, Ludwig M. Heindl1 1Department of Ophthalmology, University of Cologne The goal of this protocol is to present transcanalicular laser-assisted dacryocystorhinostomy as a minimally invasive approach in the treatment of primary acquired nasolacrimal duct obstruction. Genetics Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples Rizwan Rehimi1, Michaela Bartusel1, Francesca Solinas2, Janine Altmüller2, Alvaro Rada-Iglesias1,3 1Center for Molecular Medicine Cologne (CMMC), University of Cologne, 2Cologne Center for Genomics (CCG), University of Cologne, 3Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases (CECAD), Research Center and Systems Biology of Ageing Cologne, University of Cologne Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples. Medicine Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies Gabriel Peinkofer1,2, Juergen Hescheler2, Marcel Halbach1 1Department of Internal Medicine III, University of Cologne, 2Institute for Neurophysiology, University of Cologne Here, we describe the preparation of viable ventricular slices from adult mice and their use for sharp electrode action potential recordings. These multicellular preparations provide a preserved in vivo like tissue structure, which makes them a valuable model for electrophysiological and pharmacological studies in vitro. Developmental Biology Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders Elke Gabriel1, Jay Gopalakrishnan1,2 1Center for Molecular Medicine Cologne, University of Cologne, 2Institute for Biochemistry I, Medical School of University of Cologne Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described. Neuroscience The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster Soeren Diegelmann1, Annika Jansen1, Shreyas Jois1,2, Katharina Kastenholz1, Laura Velo Escarcena1, Nicole Strudthoff1, Henrike Scholz1 1Institute of Zoology, Albertus-Magnus University of Cologne, 2Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake. Behavior Automatic Detection of Highly Organized Theta Oscillations in the Murine EEG Ralf Müller1, Anna Papazoglou2, Julien Soos2, Andreas Lundt2, Carola Wormuth2, Christina Henseler2, Dan Ehninger3, Karl Broich4, Marco Weiergräber2 1Department of Psychiatry and Psychotherapy, University of Cologne, 2Department of Neuropsychopharmacology, Federal Institute for Drugs and Medical Devices, 3Molecular and Cellular Cognition Lab, German Center for Neurodegenerative Diseases, 4Federal Institute for Drugs and Medical Devices Theta activity in the hippocampus is related to specific cognitive and behavioral stages. Here, we describe an analytical method to detect highly-organized theta oscillations within the hippocampus using a time-frequency (i.e., wavelet analysis)-based approach. Chemistry Influence of Hybrid Perovskite Fabrication Methods on Film Formation, Electronic Structure, and Solar Cell Performance Tobias Schnier1, Jennifer Emara1, Selina Olthof1, Klaus Meerholz1 1Department of Chemistry, University of Cologne We present an extensive study on the effects of different fabrication methods for organic/inorganic perovskite thin films by comparing crystal structures, density of states, energy levels, and ultimately the solar cell performance. Biochemistry Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD Christian Pichlo1, Angelika A. Montada1, Magdalena Schacherl1, Ulrich Baumann1 1Institute of Biochemistry, University of Cologne Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein. Cancer Research The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics Juliane Mundorf1, Mirka Uhlirova1 1Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness. Medicine Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera Simona L. Schlereth1, Stefan Kremers1, Claus Cursiefen1, Ludwig M. Heindl1 1Department of Ophthalmology, University of Cologne Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used. Behavior Experimental Research Examining How People Can Cope with Uncertainty Through Soft Haptic Sensations Femke van Horen1, Thomas Mussweiler2 1Marketing Department, VU University, 2Social Psychology Department, University of Cologne To date research has focused on cognitive strategies people adopt to cope with uncertainty. This research examines instead an experiential way of dealing with uncertainty and introduces a set of experimental methods showing how the experience of haptic softness can serve as a tool to deal with uncertainty. Immunology and Infection Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1 Elena Rahn1, Katharina Thier1, Philipp Petermann1, Dagmar Knebel-Mörsdorf1,2 1Center for Biochemistry, University of Cologne, 2Department of Dermatology, University of Cologne The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin. Developmental Biology Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation Vaibhav Shinde*1, Stefanie Klima*2, Perumal Srinivasan Sureshkumar1, Kesavan Meganathan1, Smita Jagtap1, Eugen Rempel3, Jörg Rahnenführer3, Jan Georg Hengstler4, Tanja Waldmann2, Jürgen Hescheler1, Marcel Leist*2, Agapios Sachinidis*1 1Center of Physiology and Pathophysiology, Institute of Neurophysiology, University of Cologne, 2Department of Biology, University of Konstanz, 3Department of Statistics, Technical University of Dortmund, 4Leibniz Research Centre for Working Environment and Human Factors, Technical University of Dortmund The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing. Neuroscience The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern Henriette A. Seichter1, Felix Blumenthal1, Carmen R. Smarandache-Wellmann1 1Emmy Noether Group, Institute of Zoology, University of Cologne Here we describe the dissection of the crayfish abdominal nerve cord. We also demonstrate an electrophysiological technique to record fictive locomotion from swimmeret motor neurons. Immunology and Infection Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach Saleta Sierra1, Rolf Kaiser1, Nadine Lübke1, Alexander Thielen2, Eugen Schuelter1, Eva Heger1, Martin Däumer3, Stefan Reuter4, Stefan Esser5, Gerd Fätkenheuer6, Herbert Pfister1, Mark Oette7, Thomas Lengauer2 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]). Neuroscience Studying the Neural Basis of Adaptive Locomotor Behavior in Insects Matthias Gruhn1, Philipp Rosenbaum1, Hans-Peter Bollhagen1, Ansgar Bueschges1 1Zoological Institute, University of Cologne We describe a method to record motor activity, timed to the electrically recorded tarsal contact signal in a tethered insect, walking on a slippery surface. This is used to study the neural basis of adaptive behavior under reduced influence of mechanical interaction between legs through the substrate.
