University of Freiburg View Institution's Website 29 articles published in JoVE Medicine Erosion Identification in Metacarpophalangeal Joints in Rheumatoid Arthritis using High-Resolution Peripheral Quantitative Computed Tomography Yousif Al-Khoury1,2, Stephanie Finzel3, Camille Figueiredo4, Andrew J. Burghardt5, Kathryn S. Stok6, Lai-Shan Tam7, Isaac Cheng7, Justin J. Tse1, Sarah L. Manske1 1Department of Radiology, McCaig Institute for Bone and Joint Health, University of Calgary, 2Department of Biomedical Engineering, University of Calgary, 3University Medical Center Freiburg, 4Department of Rheumatology, University of São Paulo, 5Department of Radiology and Biomedical Imaging, University of California, San Francisco, 6Department of Biomedical Engineering, University of Melbourne, 7Department of Medicine and Therapeutics, The Chinese University of Hong Kong Bone erosions are an important pathological feature of rheumatoid arthritis. The purpose of this work is to introduce a training tool to provide users with guidance on identifying pathological cortical breaks on high resolution peripheral quantitative computed tomography images for erosion analysis. Biology Mesoscopic Optical Imaging of Whole Mouse Heart Francesco Giardini*1, Erica Lazzeri*1, Camilla Olianti*1, Giada Beconi1, Irene Costantini1,2, Ludovico Silvestri1,3,4, Elisabetta Cerbai1,5, Francesco S. Pavone1,3,4, Leonardo Sacconi1,3,6 1European Laboratory for Non-Linear Spectroscopy, 2Department of Biology, University of Florence, 3National Institute of Optics, National Research Council, 4Department of Physics and Astronomy, University of Florence, 5Department of Neurosciences, Psychology, Drugs and Child Health, University of Florence, 6Institute for Experimental Cardiovascular Medicine, Faculty of Medicine, University of Freiburg We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope. Behavior Animal Models of Depression - Chronic Despair Model (CDM) Stefan Vestring1,2, Tsvetan Serchov3,4, Claus Normann1,5 1Department of Psychiatry and Psychotherapy, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, 2Berta-Ottenstein-Programme for Clinician Scientists, Faculty of Medicine, University of Freiburg, 3Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, 4Department of Stereotactic and Functional Neurosurgery, Medical Center - University Freiburg, Faculty of Medicine, University of Freiburg, 5Center for Basics in Neuromodulation, Faculty of Medicine, University of Freiburg The chronic despair mouse model (CDM) of depression consists of repetitive forced swim sessions and another delayed swim phase as a read-out. It represents a suitable model for induction of a chronic depressive-like state stable for at least 4 weeks, amendable to evaluate subchronic and acute treatment interventions. Medicine Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts Laura Diaz-Maue*1,2, Janna Steinebach*1, Michael Schwaerzle8,9, Stefan Luther1,3,4,6, Patrick Ruther8,9, Claudia Richter1,5,6,7 1Research Group Biomedical Physics, Max Planck Institute for Dynamics and Self-Organization, 2Research Electronics Department, Max Planck Institute for Dynamics and Self-Organization, 3Department of Pharmacology and Toxicology, University Medical Center Goettingen, 4Institute for Nonlinear Dynamics, Georg-August-University Goettingen, 5Department of Cardiology and Pneumology, University Medical Center Goettingen, 6German Center for Cardiovascular Research, DZHK e.V., partner site Goettingen, 7Laboratory Animal Science Unit, German Primate Center Leibniz Institute for Primate Research, 8Department of Microsystems Engineering (IMTEK), University of Freiburg, 9Cluster of Excellence BrainLinks-BrainTools, University of Freiburg This work reports a method for controlling the cardiac rhythm of intact murine hearts of transgenic channelrhodopsin-2 (ChR2) mice using local photostimulation with a micro-LED array and simultaneous optical mapping of epicardial membrane potential. Biochemistry Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry Danye Qiu1, Verena B. Eisenbeis1, Adolfo Saiardi2, Henning J. Jessen1 1Institute of Organic Chemistry, University of Freiburg, 2Medical Research Council, Laboratory for Molecular Cell Biology, University College London A procedure for capillary electrophoresis electrospray ionization mass spectrometry for the absolute quantitation of inositol pyrophosphates from mammalian cell extracts is described. Chemistry Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro Andreas Schreiber*1,2, Lara G. Stühn*1,2, Süreyya E. Geissinger1,2, Matthias C. Huber1,2, Stefan M. Schiller1,2,3,4,5,6 1Center for Biological Systems Analysis, University of Freiburg, 2Faculty of Biology, University of Freiburg, 3Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, 4BIOSS Centre for Biological Signalling Studies, University of Freiburg, 5IMTEK Department of Microsystems Engineering, University of Freiburg, 6Cluster of Excellence livMatS at FIT - Freiburg Center for Interactive Materials and Bioinspired Technologies, University of Freiburg At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo. Chemistry Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy Adrianna Kolberg1, Christiane Wenzel1, Thorsten Hugel1,3, Markus Gallei2, Bizan N. Balzer1,3 1Institute of Physical Chemistry, Albert-Ludwigs-Universität Freiburg, 2Chair in Polymer Chemistry, Saarland University, 3Cluster of Excellence livMatS at FIT - Freiburg Center for Interactive Materials and Bioinspired Technologies, University of Freiburg Covalent attachment of probe molecules to atomic force microscopy (AFM) cantilever tips is an essential technique for the investigation of their physical properties. This allows us to determine the stretching force, desorption force and length of polymers via AFM-based single molecule force spectroscopy with high reproducibility. Medicine Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity Ramona A. Kopton1,2,3, Cinthia Buchmann1,2, Robin Moss1,2, Peter Kohl1,2, Rémi Peyronnet1,2, Franziska Schneider-Warme1,2 1Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg-Bad Krozingen, Medical Center-University of Freiburg, 2Faculty of Medicine, University of Freiburg, 3Faculty of Biology, University of Freiburg We present a protocol for evaluating the electromechanical effects of GtACR1 activation in rabbit cardiomyocytes. We provide detailed information on cell isolation, culturing and adenoviral transduction, and on functional experiments with the patch-clamp and carbon-fiber techniques. 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. Neuroscience Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice Jonas-Frederic Sauer1, Michael Strüber1, Marlene Bartos1 1Institute of Physiology I, University of Freiburg This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice. Biochemistry Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions Markus Götz1, Philipp Wortmann1, Sonja Schmid1,2, Thorsten Hugel1 1Institute of Physical Chemistry, University of Freiburg, 2Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions. Neuroscience Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark Patrick Bovio1,2, Deborah Roidl1, Stefanie Heidrich1, Tanja Vogel1, Henriette Franz1 1Institute for Anatomy and Cell Biology, Department of Molecular Embryology, Faculty of Medicine, University of Freiburg, 2Faculty of Biology, University of Freiburg We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3. Neuroscience Transcranial Electrical Brain Stimulation in Alert Rodents Brita Fritsch1, Anne-Kathrin Gellner1, Janine Reis1 1Department of Neurology, Albert-Ludwigs-University Freiburg This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull. Bioengineering Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation Richard Bruch*1, André Kling*1,3, Gerald A. Urban1,2, Can Dincer1,2 1Department of Microsystems Engineering, University of Freiburg, 2Freiburg Materials Research Center, University of Freiburg, 3Department of Biosystems Science and Engineering, ETH Zurich A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique. Neuroscience Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans Wolfgang Taube1, Christian Leukel1,2, Jens Bo Nielsen3,4, Jesper Lundbye-Jensen3,4 1Department of Medicine, Movement and Sport Science, University of Fribourg (Switzerland), 2Department of Sport Science, University of Freiburg (Germany), 3Department of Neuroscience and Pharmacology, University of Copenhagen, 4Department of Nutrition, Exercise and Sports, University of Copenhagen The aim of the present study was to assess changes in transmission at the corticomotoneuronal synapses in humans after repetitive transcranial magnetic stimulation. For this purpose, an electrophysiological method is introduced that allows assessment of pathway specific corticospinal transmission, i.e. differentiation of fast, direct corticospinal pathways from polysynaptic connections. Genetics From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes Tü6028 Anja Greule1, Songya Zhang1, Thomas Paululat2, Andreas Bechthold1 1Department of Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-Universität Freiburg, Germany, 2Department of Chemistry and Biology, Universität Siegen Here we present a detailed protocol of (A) the identification of a natural product with antibiotic activity, (B) the purification of the compound, (C) the first model of its biosynthesis, (D) genome sequencing/-mining and the (E) verification of the biosynthetic gene cluster. Biology LED Thermo Flow — Combining Optogenetics with Flow Cytometry Kathrin Brenker1,2,3, Kerstin Osthof1,4, Jianying Yang1,3, Michael Reth1,3,5 1Max-Planck Institute for Immunobiology und Epigenetics, 2Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, 3Centre for Biological Signaling Studies, BIOSS, University of Freiburg, 4Albert-Ludwigs-Universität, 5Institute for Biology III (Mol. Immunology), Albert-Ludwigs-Universität Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow. Behavior Force and Position Control in Humans - The Role of Augmented Feedback Benedikt Lauber1,2, Martin Keller2, Christian Leukel1,3, Albert Gollhofer1, Wolfgang Taube2 1Department of Sport Science, University of Freiburg, 2Department of Medicine, Movement and Sport Science, University of Fribourg, 3Bernsteincenter Freiburg Controlling an identical movement with position or force feedback results in different neural activation and motor behavior. This protocol describes how to investigate behavioral changes by looking at neuromuscular fatigue and how to evaluate motor cortical (inhibitory) activity using subthreshold TMS with respect to the interpretation of augmented feedback. Immunology and Infection Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice Nadine Herr1, Maximilian Mauler1,2, Christoph Bode1, Daniel Duerschmied1 1Department of Cardiology and Angiology I, Heart Center, University of Freiburg, 2Faculty of Biology, University of Freiburg This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions. Medicine Single-stage Dynamic Reanimation of the Smile in Irreversible Facial Paralysis by Free Functional Muscle Transfer Jan Thiele1, Holger Bannasch1, G. Bjoern Stark1, Steffen U. Eisenhardt1 1Department of Plastic and Hand Surgery, University of Freiburg Medical Centre The use of the masseteric nerve as donor nerve represents a single-stage alternative to the criterion standard two-stage procedure of cross-facial nerve grafting and free muscle transfer in facial paralysis. We provide a detailed description to safely perform this technique with a gracilis muscle transfer and discuss indications and limitations. Neuroscience Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types Vishal Menon1, Ria Thomas1,2, Arun R. Ghale1,3, Christina Reinhard1, Jan Pruszak1,4 1Emmy Noether-Group for Stem Cell Biology, Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, 2Spemann Graduate School of Biology and Medicine and Faculty of Biology, University of Freiburg, 3School of Life Sciences, Keele University, 4Center for Biological Signaling Studies (BIOSS), University of Freiburg We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology. Neuroscience Isolation and Culture of Mouse Cortical Astrocytes Sebastian Schildge1, Christian Bohrer1, Kristina Beck2, Christian Schachtrup1 1Institute of Anatomy and Cell Biology, University of Freiburg, 2Centre of Chronic Immunodeficiency (CCI), University Medical Centre Freiburg, University of Freiburg Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells. 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. Immunology and Infection Granulocyte-dependent Autoantibody-induced Skin Blistering Kinga Csorba1, Sebastian Sitaru2, Cassian Sitaru1,3 1Department of Dermatology, University of Freiburg, 2Kepler High School Freiburg, 3Centre for Biological Signalling Studies (BIOSS), University of Freiburg In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human epidermolysis bullosa acquisita (EBA)1. Biology Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations Dominik Schnerch1, Marie Follo1, Julia Felthaus1, Monika Engelhardt1, Ralph Wäsch1 1Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint. Medicine Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle Jan R. Thiele1, Kurt Goerendt1, G. Bjoern Stark1, Steffen U. Eisenhardt1 1Department of Plastic and Hand Surgery, University of Freiburg Medical Centre Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations. Bioengineering Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip Guido Grossmann1, Matthias Meier2,3,4, Heather N. Cartwright1, Davide Sosso1, Stephen R. Quake2,3, David W. Ehrhardt1, Wolf B. Frommer1 1Department of Plant Biology, Carnegie Institution for Science, 2Howard Hughes Medical Institute, 3Departments of Applied Physics and Bioengineering, Stanford University, 4Department of Microsystems Engineering (IMTEK) and Center for Biological Signaling Studies (BIOSS), University of Freiburg This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels. Immunology and Infection Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells Geoffrey P. Chase1, Martin Schwemmle1 1Department of Virology, Universitätsklinikum Freiburg Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively. Biology Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates Gina J. Fiala1,2,3, Wolfgang W. A. Schamel*2,3, Britta Blumenthal*3 1Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, 2Centre for Biological Signalling Studies (bioss) and Biology III, Faculty of Biology, University of Freiburg, 3Department of Molecular Immunology, Max-Planck-Institute of Immunology and Epigenetics In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.