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Max Delbrück Center for Molecular Medicine

12 ARTICLES PUBLISHED IN JoVE

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Neuroscience

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord
Hannes Schmidt 1, Fritz G. Rathjen 1
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.

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Biology

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development
Verónica A. Lombardo 1, Anje Sporbert 1, Salim Abdelilah-Seyfried 1
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.

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Biology

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle
Cécile Otten 1, Salim Abdelilah-Seyfried 1
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.

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Immunology and Infection

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging
Helmar Waiczies 1,2, Martin Guenther 1,2, Julia Skodowski 1,2, Stefano Lepore 1,2, Andreas Pohlmann 2, Thoralf Niendorf 1,2, Sonia Waiczies 1,2
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.

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Immunology and Infection

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers
Zoltan Cseresnyes *1,2, Laura Oehme *3, Volker Andresen 4, Anje Sporbert 2, 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.

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Developmental Biology

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis
Verónica A. Lombardo *1,2,3, Cécile Otten *1,2, Salim Abdelilah-Seyfried 1,2,3
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.

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Behavior

Measurement of Vibration Detection Threshold and Tactile Spatial Acuity in Human Subjects
Rabih Moshourab 1,2, Henning Frenzel 2, Stefan Lechner 3, Julia Haseleu 2, Valérie Bégay 2, Damir Omerbašić 2, Gary R. Lewin 2
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.

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JoVE Core

Cardiac Magnetic Resonance Imaging at 7 Tesla
Daniel Stäb 1,2, Aiman Al Najjar 1, Kieran O'Brien 1,3, Wendy Strugnell 4, Jonathan Richer 3, Jan Rieger 5, Thoralf Niendorf 5, Markus Barth 1
1The Centre for Advanced Imaging, The University of Queensland, Brisbane, Australia, 2Department of Diagnostic and Interventional Radiology, University Clinic Würzburg, Würzburg, Germany, 3Siemens Healthcare Pty Ltd, Brisbane, Australia, 4Richard Slaughter Centre of Excellence in CVMRI, The Prince Charles Hospital, Brisbane, Australia, 5MRI.Tools GmbH, Berlin, Germany

The sensitivity gain inherent to ultrahigh field magnetic resonance holds promise for high spatial resolution imaging of the heart. Here, we describe a protocol customized for functional cardiovascular magnetic resonance (CMR) at 7 Tesla using an advanced multi-channel radio-frequency coil, magnetic field shimming and a triggering concept.

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Medicine

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells
Thais Bascuas 1,2, Martina Kropp 1,2, Nina Harmening 1,2, Mohammed Asrih 1, Zsuzsanna Izsvák 3, Gabriele Thumann 1,2
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.

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Medicine

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System
Sandra Johnen 1, Nina Harmening 2,3, Corinne Marie 4,5, Daniel Scherman 4, Zsuzsanna Izsvák 6, Zoltán Ivics 7, Peter Walter 1, Gabriele Thumann 2,3
1Department of Ophthalmology, University Hospital RWTH Aachen, 2Experimental Ophthalmology, University of Geneva, 3Department of Ophthalmology, University Hospitals of Geneva, 4Université de Paris, CNRS, INSERM, UTCBS, Unité des technologies Chimiques et Biologiques pour la Santé, 5Chimie ParisTech, PSL Research University, 6Max Delbrück Center for Molecular Medicine in the Helmholtz Association, 7Division of Medical Biotechnology, Paul-Ehrlich-Institute

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

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Medicine

Magnetic Resonance Imaging of Multiple Sclerosis at 7.0 Tesla
Sonia Waiczies *1, Antje Els *1, Joseph Kuchling *2,3,4, Karin Markenroth Bloch 5, Anna Pankowska 6,7, Helmar Waiczies 8, Carl Herrmann 1, Claudia Chien 2,3, Carsten Finke 4,9, Friedemann Paul 2,3,4, Thoralf Niendorf 1,2,8
1Berlin Ultrahigh Field Facility (B.U.F.F.), Max Delbrück Center for Molecular Medicine in the Helmholtz Association, 2Experimental and Clinical Research Center, Max Delbrueck Center for Molecular Medicine and Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 3NeuroCure Clinical Research Center, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 4Department of Neurology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 5The Swedish National 7T Facility, Lund University Bioimaging Center, Lund University, 6Department of Radiography, Medical University of Lublin, 7ECOTECH-COMPLEX, Maria Curie-Skłodowska University, 8MRI.TOOLS GmbH, 9Berlin School of Mind and Brain, Humboldt-Universität zu Berlin

Here, we present a protocol to acquire magnetic resonance (MR) images of multiple sclerosis (MS) patient brains at 7.0 Tesla. The protocol includes preparation of the setup including the radio-frequency coils, standardized interview procedures with MS patients, subject positioning in the MR scanner and MR data acquisition.

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Biology

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy
Thais Bascuas *1,2, Martina Kropp *1,2, Nina Harmening 1,2, Brittany M. Wong 1, Sandra Johnen 3, Zsuzsanna Izsvák 4, Gabriele Thumann 1,2
1Experimental Ophthalmology, University of Geneva, 2Department of Ophthalmology, University Hospitals of Geneva, 3Department of Ophthalmology, University Hospital RWTH Aachen, 4Max Delbrück Center for Molecular Medicine

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo analyses and in vivo studies transferable to humans.

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