Ludwig Maximilian University of Munich 15 articles published in JoVE Biology Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine Jessica Radmer1, Johanna Geuder1, Fiona C. Edenhofer1, Wolfgang Enard1, Mari Ohnuki1,2,3 1Faculty of Biology, Ludwig Maximilian University of Munich, 2Institute for the Advanced Study of Human Biology, Kyoto University, 3Hakubi Center, Kyoto University The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs. Neuroscience Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis Christian Friess1, Magdalena Götz1,2,3, Jacob Kjell1,2,4 1Division of Physiological Genomics, Biomedical Center, Ludwig Maximilian University of Munich, 2Institute for Stem Cell Research, Helmholtz Zentrum München, 3SYNERGY, Excellence Cluster Systems Neurology, University of Munich, 4Department of Clinical Neuroscience, Karolinska Institutet Cryo-section-dissection allows fresh, frozen preparation of the largest neurogenic niche in the murine brain for deep quantitative proteome analysis. The method is precise, efficient, and causes minimal tissue perturbation. Therefore, it is ideally suited for studying the molecular microenvironment of this niche, as well as other organs, regions, and species. Medicine Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice Philipp Tomsits1,2,3, Kavi Raj Chataut1,2, Aparna Sharma Chivukula1,2, Li Mo1,2, Ruibing Xia1,2, Dominik Schüttler1,2,3, Sebastian Clauss1,2,3 1Department of Medicine I, University Hospital Munich, Campus Großhadern, Ludwig-Maximilians University Munich (LMU), 2DZHK (German Centre for Cardiovascular Research), Partner Site Munich, Munich Heart Alliance (MHA), 3Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians University Munich (LMU) Here we present a step-by-step protocol for a semiautomated approach to analyze murine long-term electrocardiography (ECG) data for basic ECG parameters and common arrhythmias. Data are obtained by implantable telemetry transmitters in living and awake mice and analyzed using Ponemah and its analysis modules. Medicine Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node Ruibing Xia1,2,3, Simone Loy1, Stefan Kääb1,3, Anna Titova1, Christian Schulz1,2,3, Steffen Massberg1,2,3, Sebastian Clauss1,2,3 1University Hospital Munich, Department of Medicine I, Ludwig-Maximilian-Unversity Munich (LMU), 2Insitute of Surgical Research at the Walter Brendel Center of Experimental Medicine, University Hospital Munich, Ludwig-Maximilians-University (LMU), 3German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance (MHA) The protocol presented here provides a step-by-step approach for the isolation of cardiac resident macrophages from the sinoatrial node (SAN) and atrioventricular node (AVN) region of mouse hearts. Medicine Development and Evaluation of 3D-Printed Cardiovascular Phantoms for Interventional Planning and Training Maximilian Grab1,2, Carina Hopfner3, Alena Gesenhues4, Fabian König1,2, Nikolaus A. Haas3, Christian Hagl1, Adrian Curta4, Nikolaus Thierfelder1 1Department of Cardiac Surgery, Ludwig Maximilian University Munich, 2Chair of Medical Materials and Implants, Technical University of Munich, 3Department Pediatric Cardiology and Pediatric Intensive Care, Ludwig Maximilian University Munich, 4Department of Radiology, Ludwig Maximilian University Munich Here we present development of a mock circulation setup for multimodal therapy evaluation, pre-interventional planning, and physician-training on cardiovascular anatomies. With the application of patient-specific tomographic scans, this setup is ideal for therapeutic approaches, training, and education in individualized medicine. Medicine Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse Ruibing Xia1,2,3, Julia Vlcek1,2, Julia Bauer1,2,3, Stefan Kääb1,3, Hellen Ishikawa-Ankerhold1,2, Dominic Adam van den Heuvel1,2, Christian Schulz1,2,3, Steffen Massberg1,2,3, Sebastian Clauss1,2,3 1University Hospital Munich, Department of Medicine I, Ludwig Maximilians University (LMU) Munich, 2Institute of Surgical Research at the Walter Brendel Center of Experimental Medicine, University Hospital Munich, Ludwig Maximilians University (LMU) Munich, 3German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance We provide a step-by-step protocol for whole-mount immunofluorescence staining of the sinoatrial node (SAN) and atrioventricular node (AVN) in murine hearts. Biology Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current Philipp Tomsits*1,2,3, Dominik Schüttler*1,2,3, Stefan Kääb1,2, Sebastian Clauss*1,2,3, Niels Voigt*4,5,6 1Department of Medicine I, University Hospital Munich, Campus Großhadern, Ludwig-Maximilians University Munich (LMU), 2Partner Site Munich, Munich Heart Alliance (MHA), DZHK (German Centre for Cardiovascular Research), 3Walter Brendel Center of Experimental Medicine, Ludwig-Maximilians University Munich (LMU), 4Institute of Pharmacology and Toxicology, University Medical Center Göttingen, 5Partner Site Göttingen, DZHK (German Centre for Cardiovascular Research), 6Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described. Medicine Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface Amelie Tsoutsoulopoulos1, Katrin Gohlsch2, Niklas Möhle3, Andreas Breit2, Sebastian Hoffmann5, Olaf Krischenowski3,4, Harald Mückter2, Thomas Gudermann2, Horst Thiermann1, Michaela Aufderheide3,4, Dirk Steinritz1,2 1Bundeswehr Institute of Pharmacology and Toxicology, 2Walther Straub Institute of Pharmacology and Toxicology, University of Munich, 3Cultex Laboratories GmbH, 4Cultex Technologies GmbH (formerly Cultex Laboratories GmbH), 5seh consulting + services We present a robust, transferable and predictive in vitro exposure system for the screening and monitoring of airborne particles concerning their acute pulmonary cytotoxicity by exposing cultivated human lung cells at the air-liquid interface (ALI). 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. Developmental Biology Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon Tamara Durovic1,2, Jovica Ninkovic1,3,4 1Institute of Stem Cell Research, Helmholtz Zentrum München, 2Graduate School of Systemic Neurosciences, Ludwig Maximilian University, 3Department of Cell Biology and Anatomy, Biomedical Center, Ludwig Maximilian University, 4Munich Cluster for Systems Neurology (SyNergy) Presented here is an electroporation method for plasmid DNA delivery and ependymoglial cell labeling in the adult zebrafish telencephalon. This protocol is a quick and efficient method to visualize and trace individual ependymoglial cells and opens up new possibilities to apply electroporation to a broad range of genetic manipulations. Medicine Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses Carina Schuster1, Sebastian Sterz1, Daniel Teupser1, Mathias Brügel1, Michael Vogeser1, Michael Paal1 1Institute of Laboratory Medicine, University Hospital, LMU Munich Here we present a tandem mass spectrometry-based protocol for the quantification of frequently used antibiotics in intensive care units, namely cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid, and piperacillin. Cancer Research Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma Jan Hagemann*1, Christian Jacobi*2, Sabine Gstoettner3, Christian Welz4, Sabina Schwenk-Zieger3, Roland Stauber1, Sebastian Strieth1, Julian Kuenzel1, Philipp Baumeister3, Sven Becker1,3 1Department of Otorhinolaryngology, Johannes-Gutenberg University Medical Center, 2Department of Otorhinolaryngology, Technical University of Munich Medical Center, 3Department of Otorhinolaryngology, Ludwig-Maximilian-University Medical Center, 4Department of Otorhinolaryngology, University of Goettingen Medical Center We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future. Behavior Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila Sara Batelli1, Malte Kremer1,2, Christophe Jung1, Ulrike Gaul1 1Gene Center and Department of Biochemistry, Ludwig-Maximilians-University Munich, 2Janelia Farm Research Campus, Howard Hughes Medical Institute Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system. Neuroscience Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons Marisa Karow1, Christian Schichor2, Ruth Beckervordersandforth3, Benedikt Berninger1,4 1Department of Physiological Genomics, Institute of Physiology, Ludwig Maximilians University Munich, 2Tumor Biology Lab, Neurosurgical Clinic, Ludwig-Maximilians University Munich, 3Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, 4Institute of Physiological Chemistry and Focus Program Translational Neuroscience, Johannes Gutenberg University Mainz Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1. Biology Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture Bernd Rädle*1, Andrzej J. Rutkowski*2, Zsolt Ruzsics1, Caroline C. Friedel3, Ulrich H. Koszinowski1, Lars Dölken2 1Max von Pettenkofer Institute, 2Department of Medicine, University of Cambridge, 3Institute for Informatics, Ludwig-Maximilians-University Munich Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.