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University Medical Center Hamburg-Eppendorf

18 ARTICLES PUBLISHED IN JoVE

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Bioengineering

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)
Lenard Conradi 1,2, Christiane Pahrmann 1, Stephanie Schmidt 1, Tobias Deuse 1,3, Arne Hansen 2, Alexandra Eder 2, Hermann Reichenspurner 1, Robert C. Robbins 3, Thomas Eschenhagen 2, Sonja Schrepfer 1,3
1Transplant and Stem Cell Immunobiology Lab (TSI) and CVRC, University Hospital Hamburg, University Heart Center Hamburg, 2Department of Experimental and Clinical Pharmacology and Toxicology, University Heart Center Hamburg, 3CT Surgery, Stanford University School of Medicine

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

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Biology

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors
Julia U. Sprenger 1, Ruwan K. Perera 1, Konrad R. Götz 1, Viacheslav O. Nikolaev 1
1Emmy Noether Group of the DFG, Department of Cardiology and Pneumology, European Heart Research Insitute Göttingen, Georg August University Medical Center, Göttingen, Germany

Förster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

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Immunology and Infection

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus
Christina Stoeckle 1,2, Ioanna A. Rota 1, Eva Tolosa 3, Christoph Haller 4, Arthur Melms 5, Eleni Adamopoulou 1
1Department of General Neurology, Hertie Institute for Clinical Brain Research, 2Institute of Pharmacology, University of Bern, 3Department of Immunology, University Medical Center Hamburg-Eppendorf, 4Department of Thoracic and Cardiovascular Surgery, University Clinic Tuebingen, 5Department of Neurology, University Hospital Erlangen

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

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Immunology and Infection

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions
Manuel Wolters 1, Bernd Zobiak 2, Theresa Nauth 1, Martin Aepfelbacher 1
1Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, 2UKE Microscopy Imaging Facility, University Medical Center Hamburg-Eppendorf

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

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Medicine

Assessing Specificity of Anticancer Drugs In Vitro
Lan Kluwe 1,2,3
1Department of Maxillofacial Surgery, University Medical Center Hamburg-Eppendorf, 2Department of Neurology, University Medical Center Hamburg-Eppendorf, 3Laboratory for Tumor Biology and Regenerative Medicine, University Medical Center Hamburg-Eppendorf

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

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JoVE Core

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening
Ingra Mannhardt 1, Umber Saleem 1, Anika Benzin 1, Thomas Schulze 1, Birgit Klampe 1, Thomas Eschenhagen 1, Arne Hansen 1
1Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf and DZHK (German Center for Cardiovascular Research)

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

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Biochemistry

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis
Kathrin Rösch 1, Marcel Kwiatkowski 2, Hartmut Schlüter 2, Eva Herker 1
1Heinrich Pette Institute, Leibniz Institute for Experimental Virology, 2Core Facility Mass Spectrometric Proteomics, University Medical Center Hamburg-Eppendorf

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

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Cancer Research

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization
Shukun Chen *1, Amin El-Heliebi *1, Julia Schmid 1, Karl Kashofer 2, Zbigniew T. Czyż 3, Bernhard Michael Polzer 3, Klaus Pantel 4, Thomas Kroneis 1,5, Peter Sedlmayr 1
1Institute of Cell Biology, Histology and Embryology, Medical University of Graz, 2Institute of Pathology, Medical University of Graz, 3Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, 4Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, 5Sahlgrenska Cancer Center, University of Gothenburg

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

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Medicine

Invasive Hemodynamic Monitoring of Aortic and Pulmonary Artery Hemodynamics in a Large Animal Model of ARDS
Rahel Kluttig 1, Till Friedheim 1, Christoph Behem 1, Niko Zach 1, Rosannis Brown 1, Michael Graessler 1, Daniel Reuter 1, Christian Zöllner 1, Constantin Trepte 1
1Universitatsklinikum Hamburg-Eppendorf

We present a protocol of creating right ventricular dysfunction in a pig model by inducing ARDS. We demonstrate invasive monitoring of left and right ventricular cardiac output using flow probes around the aorta and the pulmonary artery, as well as blood pressure measurements in the aorta and pulmonary artery.

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Medicine

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System
Christiane Jungen 1,2, Katharina Scherschel 1,2, Nadja I. Bork 2,3, Pawel Kuklik 1, Christian Eickholt 1, Helge Kniep 1, Niklas Klatt 1,2, Stephan Willems 1,2, Viacheslav O. Nikolaev 2,3, Christian Meyer 1,2
1Department of Cardiology-Electrophysiology, cNEP (cardiac Neuro- and Electrophysiology research group), University Heart Center, University Hospital Hamburg-Eppendorf, 2DZHK (German Center for Cardiovascular Research), 3Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf

Here, we present a protocol for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cAMP dynamics using an ex vivo Langendorff setup.

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Biochemistry

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins
Eva Königshausen 1, Sebastian A. Potthoff 1, Raphael Haase 1, Catherine Meyer-Schwesinger 2, Ernest Kaufmann 1, L. Christian Rump 1, Johannes Stegbauer 1, Lorenz Sellin 1, Ivo Quack 1, Magdalena Woznowski 1
1Department of Nephrology, Medical Faculty, Heinrich-Heine-University, 2Institute of Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

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Immunology and Infection

CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites
Julia K. Bialek *1,2, Thomas Walther *1, Joachim Hauber 1,3, Ulrike C. Lange 1,2,3
1Heinrich Pette Institute, Leibniz Institute for Experimental Virology, 2Department of Anesthesiology, University Medical Center Hamburg-Eppendorf, 3German Center for Infection Research (DZIF)

We present a genome engineering workflow for the generation of new in vitro models for HIV-1 infection that recapitulate proviral integration at selected genomic sites. Targeting of HIV-derived reporters is facilitated by CRISPR-Cas9-mediated, site-specific genome manipulation. Detailed protocols for single-cell clone generation, screening, and correct targeting verification are provided.

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Bioengineering

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy
Bernd Zobiak 1, Antonio Virgilio Failla 1
1UKE Microscopy Imaging Facility, University Medical Center Hamburg-Eppendorf

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

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Medicine

Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model
Liesa Castro 1,2, Birgit Geertz 3, Marina Reinsch 2,3, Bülent Aksehirlioglu 3, Arne Hansen 2,3, Thomas Eschenhagen 2,3, Hermann Reichenspurner 1,2, Florian Weinberger *2,3, Simon Pecha *1,2
1Department of Cardiovascular Surgery, University Heart Center Hamburg, 2partner site Hamburg/Kiel/Lübeck, German Centre for Cardiovascular Research (DZHK), 3Department of Experimental Pharmacology and Toxicology, Cardiovascular ResearchCenter, University Medical Center Hamburg-Eppendorf

Here we present a protocol for the induction of left ventricular cryoinjury followed by the implantation of a cardiac muscle patch, derived from human iPS-cell cardiomyocytes in a guinea pig model.

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Developmental Biology

Magnetic Adjustment of Afterload in Engineered Heart Tissues
Benjamin Becker *1,2, Marita L. Rodriguez *1,2, Tessa R. Werner 1,2, Justus Stenzig 1,2, Thomas Eschenhagen 1,2, Marc N. Hirt 1,2
1Institute of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, 2DZHK (German Centre for Cardiovascular Research)

This protocol provides detailed methods describing the fabrication and implementation of a magnetics-based afterload tuning platform for engineered heart tissues.

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Medicine

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion
Christoph R. Behem 1, Till Friedheim 1, Sabine H. Wipper 2, Hans O. Pinnschmidt 3, Michael F. Graessler 1, Catharina Gaeth 4, Hannes Holthusen 1, Adina Rapp 5, Timo Suntrop 1, Josephina Haunschild 6, Christian D. Etz 6, Constantin J. C. Trepte 1
1Department of Anesthesiology, Center of Anesthesiology and Intensive Care Medicine, University Medical Center Hamburg-Eppendorf, 2University Department for Vascular Surgery and Department of Operative Medicine, Medical University of Innsbruck, 3Department of Medical Biometry and Epidemiology, University Medical Center Hamburg-Eppendorf, 4Department of Vascular Medicine, University Heart and Vascular Center Hamburg (UHZ), 5Department of Cardiology, Rostock University Medical Center, 6University Department for Cardiac Surgery, Heart Center Leipzig

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

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Biology

Chromatin Extraction from Frozen Chimeric Liver Tissue for Chromatin Immunoprecipitation Analysis
Andrea Pirosu 1,3, Lena Allweiss 1, Maura Dandri 1,2
1Department of Internal Medicine, University Medical Center Hamburg-Eppendorf, 2German Center for Infection Research (DZIF), Hamburg-Lübeck-Borstel-Riems site, 3Research Department Virus Immunology, Leibniz Institute for Experimental Virology

This protocol focuses on chromatin preparation from snap frozen tissues and it is suitable for Crosslinking Chromatin Immunoprecipitation (X-ChIP) followed by either quantitative PCR analysis (X-ChIP-qPCR) or next generation sequencing approaches (X-ChIP-seq).

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Medicine

Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates
Hans Christian Schmidt 1,2,3, Johanna Hagens 1,2,3, Pauline Schuppert 1,2, Clara Philippi 1,2, Konrad Reinshagen 1,2, Christian Tomuschat 1,2
1Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf, 2Research Laboratory for Pediatric Surgery, 3Research Animal Facility, University Medical Center Hamburg-Eppendorf

For the observation of murine neonatal bile duct disorders, an intact bile duct and efficient preparation are required. Therefore, a new approach for isolating the entire extrahepatic bile duct system in murine neonates was successfully developed while maintaining the integrity of the bile duct.

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