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Max Delbrück Center for Molecular Medicine in the Helmholtz Association

7 ARTICLES PUBLISHED IN JoVE

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 Pohlmann², 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.

JoVE Core

Cardiac Magnetic Resonance Imaging at 7 Tesla

Daniel Stäb^1,2, Aiman Al Najjar¹, Kieran O'Brien^1,3, Wendy Strugnell⁴, Jonathan Richer³, Jan Rieger⁵, Thoralf Niendorf⁵, Markus Barth¹

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.

Developmental Biology

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Ena Kolundzic¹, Stefanie Seelk¹, Baris Tursun¹

¹Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

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 Asrih¹, Zsuzsanna Izsvák³, 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.

Medicine

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Sandra Johnen¹, Nina Harmening^2,3, Corinne Marie^4,5, Daniel Scherman⁴, Zsuzsanna Izsvák⁶, Zoltán Ivics⁷, Peter Walter¹, 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).

Medicine

Magnetic Resonance Imaging of Multiple Sclerosis at 7.0 Tesla

Sonia Waiczies^*1, Antje Els^*1, Joseph Kuchling^*2,3,4, Karin Markenroth Bloch⁵, Anna Pankowska^6,7, Helmar Waiczies⁸, Carl Herrmann¹, 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.

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. Wong¹, Sandra Johnen³, Zsuzsanna Izsvák⁴, 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.