Max-Delbrück-Center for Molecular Medicine View Institution's Website 14 articles published in JoVE Medicine Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells Thais Bascuas1,2, Martina Kropp1,2, Nina Harmening1,2, Mohammed Asrih1, Zsuzsanna Izsvák3, Gabriele Thumann1,2 1Experimental Ophthalmology, University of Geneva, 2Department of Ophthalmology, University Hospitals of Geneva, 3Max Delbrück Center for Molecular Medicine We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration. Genetics Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry Sarah E. Ashley1,2, Braydon A. Meyer2,3, Justine A. Ellis2,3,4, David J. Martino2,3,5 1Molecular Genetics of Chronic Inflammation and Allergic Disease, Max-Delbrück Center for Molecular Medicine, 2 Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies. 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. Developmental Biology Automated Quantification of Hematopoietic Cell – Stromal Cell Interactions in Histological Images of Undecalcified Bone Sandra Zehentmeier1, Zoltan Cseresnyes2,3, Juan Escribano Navarro4, Raluca A. Niesner2, Anja E. Hauser1,5 1Immunodynamics, German Rheumatism Research Center, a Leibniz Institute, 2Biophysical Analytics, German Rheumatism Research Center, a Leibniz Institute, 3Max-Delbrück Center for Molecular Medicine, 4Wimasis GmbH, 5Immunodynamics and Intravital Imaging, Charité - University of Medicine A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions. Developmental Biology Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis Verónica A. Lombardo*1,2,3, Cécile Otten*1,2, Salim Abdelilah-Seyfried1,2,3 1Max Delbrück Center for Molecular Medicine, 2Institute of Biochemistry and Biology, University of Potsdam, 3Institute of Molecular Biology, Medizinische Hochschule Hannover To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA. Immunology and Infection Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers Zoltan Cseresnyes*1,2, Laura Oehme*3, Volker Andresen4, Anje Sporbert2, Anja E. Hauser*3,5, Raluca Niesner*1 1Biophysical Analytics, German Rheumatism Research Center, Leibniz Institute, 2Microscopy Core Facility, Max-Delbrück Center for Molecular Medicine, 3Immunodynamics, German Rheumatism Research Center, Leibniz Institute, 4LaVision Biotec GmbH, 5Immunodynamics and Intravital Imaging, Charité - University of Medicine High-resolution intravital imaging with enhanced contrast up to 120 µm depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 µm depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits. Biology Culturing Primary Rat Inner Medullary Collecting Duct Cells Dörte Faust1, Andrea Geelhaar1, Beate Eisermann1, Jenny Eichhorst2, Burkhard Wiesner2, Walter Rosenthal1,3, Enno Klussmann1 1Anchored Signalling, Max-Delbrück-Center for Molecular Medicine, 2Leibniz Institute for Molecular Pharmacology (FMP), 3Charité University Medicine Berlin Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption. Immunology and Infection Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging Helmar Waiczies1,2, Martin Guenther1,2, Julia Skodowski1,2, Stefano Lepore1,2, Andreas Pohlmann2, Thoralf Niendorf1,2, Sonia Waiczies1,2 1Experimental and Clinical Research Center, A joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine, 2Berlin Ultrahigh Field Facility (B.U.F.F.), Max Delbrück Center for Molecular Medicine Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS. Biology Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes Clemens Köhncke1,2, Ulrike Lisewski1, Leonhard Schleußner1, Carolin Gaertner1, Saskia Reichert1, Torsten K. Roepke1,3 1Experimental and Clinical Research Center (ECRC), Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine (MDC), 2Medical Department, Division of Cardiology, Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, 3Medical Department, Division of Cardiology and Angiology, Campus Mitte, Charité - Universitätsmedizin Berlin Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays. Biology Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle Cécile Otten1, Salim Abdelilah-Seyfried1 1Max Delbrück Center for Molecular Medicine The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage. Biology Cell Tracking Using Photoconvertible Proteins During Zebrafish Development Verónica A. Lombardo1, Anje Sporbert1, Salim Abdelilah-Seyfried1 1Max Delbrück Center for Molecular Medicine Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism. Biology A System for ex vivo Culturing of Embryonic Pancreas Kristin M. Petzold1, Francesca M. Spagnoli1 1Molecular and Cellular Basis of Embryonic Development, Max-Delbrück-Center for Molecular Medicine Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging. Neuroscience DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord Hannes Schmidt1, Fritz G. Rathjen1 1Developmental Neurobiology, Max Delbrück Center for Molecular Medicine The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons. Biology PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins Markus Hafner1, Markus Landthaler2, Lukas Burger3, Mohsen Khorshid3, Jean Hausser4, Philipp Berninger4, Andrea Rothballer1, Manuel Ascano1, Anna-Carina Jungkamp2, Mathias Munschauer2, Alexander Ulrich1, Greg S. Wardle1, Scott Dewell5, Mihaela Zavolan3, Thomas Tuschl1 1Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, Rockefeller University, 2Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, 3Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 4Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 5Genomics Resource Center, Rockefeller University RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.