University of Heidelberg View Institution's Website 29 articles published in JoVE Immunology and Infection Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues Lavinia Schoenfeld1, Birgit Appl1, Laia Pagerols-Raluy1, Annika Heuer3, Konrad Reinshagen1, Michael Boettcher1,2 1Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf, University of Hamburg, 2Department of Pediatric Surgery, University Medical Center Mannheim, University of Heidelberg, 3Division of Spine Surgery, Department of Trauma and Orthopedic Surgery, University Medical Center Hamburg-Eppendorf (UKE) Neutrophil extracellular traps (NETs) are associated with various diseases, and immunofluorescence is often used for their visualization. However, there are various staining protocols, and, in many cases, only one type of tissue is examined. Here, we establish a generally applicable protocol for staining NETs in mouse and human tissue. Bioengineering Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus (AAV) Capsid Variants Kleopatra Rapti1,2, Olena Maiakovska1,2, Jonas Becker1,2, Joanna Szumska1,2, Margarita Zayas1,2, Felix Bubeck1,2, Jixin Liu1,2, Emma Gerstmann1,2, Chiara Krämer1,2, Ellen Wiedtke1,2, Dirk Grimm1,2,3,4,5 1Department of Infectious Diseases/Virology, Section Viral Vector Technologies, Medical Faculty, University of Heidelberg, 2BioQuant Center, BQ0030, University of Heidelberg, 3Cluster of Excellence CellNetworks, 4German Center for Infection Research (DZIF), 5German Center for Cardiovascular Research (DZHK) AAV peptide display library generation and subsequent validation through the barcoding of candidates with novel properties for the creation of next-generation AAVs. Immunology and Infection Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples Dario L. Frey*1,2, Matteo Guerra*1,2,3,4, Marcus A. Mall1,2,5,6,7, Carsten Schultz1,3,8 1Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), 2Dept. of Translational Pulmonology, University of Heidelberg, 3Molecular Medicine Partnership Unit (MMPU), European Molecular Biology Laboratory (EMBL), University of Heidelberg, 4Faculty of Biosciences, Collaboration for Joint Ph.D. Degree between EMBL and Heidelberg University, University of Heidelberg, 5Dept. of Pediatric Pulmonology, Immunology and Critical Care Medicine, Charité – Universitätsmedizin Berlin, 6Berlin Institute of Health (BIH), 7German Center for Lung Research (DZL), Associated Partner Site, Berlin, 8Dept. of Chemical Physiology and Biochemistry, Oregon Health and Science University The protocols herein described provide a guide to visualize and quantify the activity of neutrophil proteases in human sputum. The applications of such analysis span from the evaluation of anti-inflammatory treatments, to biomarker validation, drug screening and large cohort clinical studies. Biology A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision Kaixuan Yan1,2, Jiaxing Zheng1,2, Frank G. Zöllner3,4, Kay Schwenke1, Prama Pallavi1,2, Michael Keese1,2 1Department of Surgery, Medical Faculty Manheim, Heidelberg University, 2European Center of Angioscience ECAS, Medical Faculty Manheim, Heidelberg University, 3Computer-Assisted Clinical Medicine, Mannheim Institute for Intelligent Systems in Medicine, Medical Faculty Mannheim, Heidelberg University, 4Cooperative Core Facility Animal Scanner ZI, Medical Faculty Mannheim, Heidelberg University This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades. Cancer Research Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq Ashwin Narayanan1, Enrique Blanco-Carmona2, Engin Demirdizen1, Xueyuan Sun1,3, Christel Herold-Mende4, Matthias Schlesner2, Sevin Turcan1 1Neurology Clinic and National Center for Tumor Diseases, University Hospital Heidelberg, 2Bioinformatics and Omics Data Analytics, German Cancer Research Center (DKFZ), 3Clinical Cooperation Unit Neurooncology, German Cancer Research Center (DKFZ), 4Division of Experimental Neurosurgery, Department of Neurosurgery, University of Heidelberg, INF 400, Heidelberg, Germany Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. We developed a simple and efficient protocol that utilizes a combination of buffers and gradient centrifugation to isolate single nuclei from fresh frozen glioma tissues for single nucleus RNA and ATAC sequencing studies. Biology A High Output Method to Isolate Cerebral Pericytes from Mouse Anupriya Mehra1, Lucie Dehouck1, Elodie Vandenhaute1, Marc Fatar2, Laurence Fenart1, Fabien Gosselet1 1Laboratory of the Blood Brain Barrier, University of Artois, 2Department of Neurology, Universitätsmedizin Mannheim, Heidelberg University We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used. Immunology and Infection Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry Guido Wabnitz1, Henning Kirchgessner1, Yvonne Samstag1 1Institute of Immunology, Section Molecular Immunology, University of Heidelberg Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time. Bioengineering Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells Rachel Santarella-Mellwig1, Uta Haselmann2, Nicole L. Schieber1, Paul Walther3, Yannick Schwab1, Claude Antony1, Ralf Bartenschlager2,4, Inés Romero-Brey2 1European Molecular Biology Laboratory, 2Department of Infectious Diseases, Molecular Virology, Heidelberg University, 3Central Facility for Electron Microscopy, Ulm University, 4Heidelberg Partner Site, German Center for Infection Research A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions. Biochemistry Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes Katharina Kohl1, Thomas Fleming2,3, Kübra Acunman1, Hans-Peter Hammes1,4, Michael Morcos*1, Andrea Schlotterer*1 15th Medical Department, Medical Faculty Mannheim, Heidelberg University, 2Department of Internal Medicine, Heidelberg University, 3German Center for Diabetes Research (DZD), 4European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture. Biochemistry Split-BioID — Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment Isabel M. Schopp1,2, Julien Béthune1,2 1Cluster of excellence CellNetworks, Heidelberg University, 2Heidelberg University Biochemistry Center (BZH) We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment. Medicine Application of an Amplitude-integrated EEG Monitor (Cerebral Function Monitor) to Neonates Nora Bruns1, Susanne Blumenthal1, Irmgard Meyer1, Susanne Klose-Verschuur1, Ursula Felderhoff-Müser1, Hanna Müller1,2 1Department of Pediatrics I, Neonatology, Pediatric Intensive Care, Pediatric Neurology, University Hospital Essen, University Duisburg-Essen, 2Division of Neonatology, Department of Pediatrics, University of Heidelberg Here, we show how to apply amplitude-integrated electroencephalography to monitor cerebral function in neonates. Cancer Research A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer Alexander M. Betzler1, Susan Kochall1, Linda Blickensdörfer2, Sebastian A. Garcia1, May-Linn Thepkaysone1, Lahiri K. Nanduri1, Michael H. Muders3, Jürgen Weitz1,4,5, Christoph Reissfelder1, Sebastian Schölch1,4,5 1Department of Gastrointestinal, Thoracic and Vascular Surgery, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, 2Department of General, Gastrointestinal and Transplant Surgery, University of Heidelberg, 3Department of Pathology, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, 4German Cancer Consortium (DKTK), 5German Cancer Research Center (DKFZ) A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented. Biology A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry Konstantin D. Bergmeister1,3, Marion Gröger2, Martin Aman1, Anna Willensdorfer1, Krisztina Manzano-Szalai1, Stefan Salminger1, Oskar C. Aszmann1 1CD Laboratory for the Restoration of Extremity Function, Division of Plastic and Reconstructive Surgery, Department of Surgery, Medical University of Vienna, 2Core Facility Imaging, Core Facilities, Medical University Vienna, 3Department of Hand, Plastic and Reconstructive Surgery, Burn Center, BG Trauma Center Ludwigshafen, Plastic and Hand Surgery, University of Heidelberg Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ. Developmental Biology In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences Stefanie Dudczig1,2, Peter D. Currie2, Lucia Poggi3, Patricia R. Jusuf1,2 1School of Biosciences, The University of Melbourne, 2Australian Regenerative Medicine Institute (ARMI), Monash University, 3The David J Apple Center for Vision Research, Department of Ophthalmology, Heidelberg University Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose. Behavior Measurement of Vibration Detection Threshold and Tactile Spatial Acuity in Human Subjects Rabih Moshourab1,2, Henning Frenzel2, Stefan Lechner3, Julia Haseleu2, Valérie Bégay2, Damir Omerbašić2, Gary R. Lewin2 1Department of Anesthesiology and Intensive Care Medicine, Charite Universitätsmedzin, Campus Virchow Klinikum und Campus Charite Mitte, 2Department of Neuroscience, Molecular Physiology of Somatic Sensation, Max Delbrück Center for Molecular Medicine, 3Institute of Pharmacology, University of Heidelberg Here, we present protocols to determine vibration detection thresholds and tactile acuity using psychophysical methods in man. Biology Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography Mila M. Leuthold1,2, Anna D. Koromyslova1,2, Bishal K. Singh1,2, Grant S. Hansman1,2 1Schaller Research Group, University of Heidelberg and the German Cancer Research Center, 2Department of Infectious Diseases, Virology, University of Heidelberg Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp). Developmental Biology Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System Carlo A. Beretta1,2,3, Nicolas Dross2, Ulrike Engel2, Matthias Carl1 1Medical Faculty Mannheim, Department of Cell and Molecular Biology, Heidelberg University, 2COS and Nikon Imaging Center at the University of Heidelberg, Heidelberg University, 3Excellenzcluster CellNetworks, University of Heidelberg We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system. Biology Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice Sebastian Uhl1, Marc Freichel1, Ilka Mathar1 1Pharmakologisches Institut, Universität Heidelberg Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics. Medicine Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery Gemma Llovera*1,2, Stefan Roth*1,2, Nikolaus Plesnila1,2, Roland Veltkamp3,4, Arthur Liesz1,2 1Institute for Stroke and Dementia Research, University Hospital Munich, 2Munich Cluster for Systems Neurology (SyNergy), 3Department of Neurology, University Heidelberg, 4Imperial College, Charing Cross Hospital Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes. Medicine A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology Christian Erbel*1, Deniz Okuyucu*1, Mohammadreza Akhavanpoor1, Li Zhao1, Susanne Wangler1, Maani Hakimi2, Andreas Doesch1, Thomas J. Dengler3, Hugo A. Katus1, Christian A. Gleissner1 1Department of Cardiology, University of Heidelberg, 2Department of Vascular Surgery, University of Heidelberg, 3Department of Cardiology, SLK Hospital am Plattenwald Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods. Biology In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice Christian Njoo1, Celine Heinl1, Rohini Kuner1 1Institute for Pharmacology, University of Heidelberg This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia. Chemistry Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry Nikolai Hentze1, Matthias P. Mayer1 1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), University of Heidelberg Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects. Chemistry Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach Theresa L. M. Pohl1, Elisabeth H. Schwab1, Elisabetta A. Cavalcanti-Adam1,2 1Department of Biophysical Chemistry, Institute for Physical Chemistry, University of Heidelberg, 2Max Planck Institute for Intelligent Systems at Stuttgart We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane. Neuroscience Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation Roberta Haddad-Tóvolli*1, Nora-Emöke Szabó*2, Xunlei Zhou1, Gonzalo Alvarez-Bolado1 1Institute of Anatomy and Cell Biology, University of Heidelberg, 2Institut de recherches cliniques de Montreal Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection. Biology Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents Niels Voigt*1,2, Xiao-Bo Zhou*2, Dobromir Dobrev1,2 1Institute of Pharmacology, University of Duisburg-Essen, 2Division of Experimental Cardiology, University of Heidelberg We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies. Immunology and Infection An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization Christian Erbel1, Gregor Rupp1, Christian M. Helmes1, Mirjam Tyka1, Fabian Linden1, Andreas O. Doesch1, Hugo A. Katus1, Christian A. Gleissner1 1Department of Cardiology, University of Heidelberg Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies. Neuroscience Analyzing Murine Schwann Cell Development Along Growing Axons Stephan Heermann1,2, Kerstin Krieglstein1,3 1Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, 2Department of Neuroanatomy, University of Heidelberg, 3FRIAS, University of Freiburg Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons. Neuroscience Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth Isabel Brachmann1, Kerry L. Tucker1 1Interdisciplinary Center for Neurosciences, Institute of Anatomy and Cell Biology, University of Heidelberg We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth. Biology Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators Martin S Angst1, Martha Tingle1, Martin Schmelz2,3, Brendan Carvalho1, David C Yeomans1 1Department of Anesthesia, Stanford University School of Medicine, 2Department of Anaesthesiology, University of Mannheim, 3Department of Anaesthesiology, University of Heidelberg A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation.