University Medical Center Hamburg Eppendorf 19 articles published in JoVE Immunology and Infection Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues Lavinia Schoenfeld1, Birgit Appl1, Laia Pagerols-Raluy1, Annika Heuer3, Konrad Reinshagen1, Michael Boettcher1,2 1Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf, University of Hamburg, 2Department of Pediatric Surgery, University Medical Center Mannheim, University of Heidelberg, 3Division of Spine Surgery, Department of Trauma and Orthopedic Surgery, University Medical Center Hamburg-Eppendorf (UKE) Neutrophil extracellular traps (NETs) are associated with various diseases, and immunofluorescence is often used for their visualization. However, there are various staining protocols, and, in many cases, only one type of tissue is examined. Here, we establish a generally applicable protocol for staining NETs in mouse and human tissue. Medicine Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates Hans Christian Schmidt1,2,3, Johanna Hagens1,2,3, Pauline Schuppert1,2, Clara Philippi1,2, Konrad Reinshagen1,2, Christian Tomuschat1,2 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. Biology Chromatin Extraction from Frozen Chimeric Liver Tissue for Chromatin Immunoprecipitation Analysis Andrea Pirosu1,3, Lena Allweiss1, Maura Dandri1,2 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). Medicine Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion Christoph R. Behem1, Till Friedheim1, Sabine H. Wipper2, Hans O. Pinnschmidt3, Michael F. Graessler1, Catharina Gaeth4, Hannes Holthusen1, Adina Rapp5, Timo Suntrop1, Josephina Haunschild6, Christian D. Etz6, Constantin J. C. Trepte1 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. Developmental Biology Magnetic Adjustment of Afterload in Engineered Heart Tissues Benjamin Becker*1,2, Marita L. Rodriguez*1,2, Tessa R. Werner1,2, Justus Stenzig1,2, Thomas Eschenhagen1,2, Marc N. Hirt1,2 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. Medicine A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons Daniel A. Wenzel1, Berenike C. Kunzmann2, Nils A. Steinhorst1, Martin S. Spitzer1, Maximilian Schultheiss1 1Department of Ophthalmology, University Medical Center Hamburg-Eppendorf (UKE), 2University Eye Hospital, Center For Ophthalmology, University Hospital Tübingen Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran. Cancer Research Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy Jane Harmer1, Nina Struve2, Anke Brüning-Richardson1 1School of Applied Sciences, University of Huddersfield, 2Laboratory of Radiotherapy and Experimental Radiation Oncology, Humbertus Wald Tumorzentrum, University Cancer Center Hamburg, University Medical Center Hamburg-Eppendorf The effects of migrastatic inhibitors on glioma cancer cell migration in three-dimensional (3D) invasion assays using a histone deacetylase (HDAC) inhibitor are characterized by high-resolution confocal microscopy. Medicine Optical Clearing and Imaging of Immunolabeled Kidney Tissue Turgay Saritas1, Victor G. Puelles1,2,3, Xiao-Tong Su4, David H. Ellison4,5,6, Rafael Kramann1,7 1Division of Nephrology and Clinical Immunology, University Hospital RWTH Aachen, 2III. Department of Medicine, University Medical Center, Hamburg-Eppendorf, 3Department of Nephrology, Monash Health, 4Division of Nephrology and Hypertension, Oregon Health and Science University, 5Renal Section, Veterans Affairs Portland Health Care System, 6Fondation LeDucq Transatlantic Networks of Excellence, 7Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures. Medicine Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model Liesa Castro1,2, Birgit Geertz3, Marina Reinsch2,3, Bülent Aksehirlioglu3, Arne Hansen2,3, Thomas Eschenhagen2,3, Hermann Reichenspurner1,2, 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. Bioengineering Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy Bernd Zobiak1, Antonio Virgilio Failla1 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. Medicine Invasive Hemodynamic Monitoring of Aortic and Pulmonary Artery Hemodynamics in a Large Animal Model of ARDS Rahel Kluttig1, Till Friedheim1, Christoph Behem1, Niko Zach1, Rosannis Brown1, Michael Graessler1, Daniel Reuter1, Christian Zöllner1, Constantin Trepte1 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. Medicine Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System Christiane Jungen1,2, Katharina Scherschel1,2, Nadja I. Bork2,3, Pawel Kuklik1, Christian Eickholt1, Helge Kniep1, Niklas Klatt1,2, Stephan Willems1,2, Viacheslav O. Nikolaev2,3, Christian Meyer1,2 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. Cancer Research Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization Shukun Chen*1, Amin El-Heliebi*1, Julia Schmid1, Karl Kashofer2, Zbigniew T. Czyż3, Bernhard Michael Polzer3, Klaus Pantel4, Thomas Kroneis1,5, Peter Sedlmayr1 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). Biochemistry Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis Kathrin Rösch1, Marcel Kwiatkowski2, Hartmut Schlüter2, Eva Herker1 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. Behavior Psychophysically-anchored, Robust Thresholding in Studying Pain-related Lateralization of Oscillatory Prestimulus Activity Philipp Taesler1, Michael Rose1 1Department of Systems Neuroscience, University Medical Center Hamburg Eppendorf Psychophysical methods such as the QUEST estimation procedure can efficiently yield robust estimates of the stimulation intensity at which nonpainful sensations transition into painful sensations. By stimulating repeatedly at the threshold intensity, the variability in rating responses can directly be attributed to perceptual classifications in subsequent analyses. Medicine Assessing Specificity of Anticancer Drugs In Vitro Lan Kluwe1,2,3 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. Immunology and Infection Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions Manuel Wolters1, Bernd Zobiak2, Theresa Nauth1, Martin Aepfelbacher1 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. Medicine Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy Peter Bannas*1, Alexander Lenz*1,2, Valentin Kunick1,2, William Fumey1,2, Björn Rissiek2, Joanna Schmid1,2, Friedrich Haag2, Axel Leingärtner3, Martin Trepel4, Gerhard Adam1, Friedrich Koch-Nolte2 1Department of Diagnostic and Interventional Radiology, University Medical Center, Hamburg, 2Institute of Immunology, University Medical Center, Hamburg, 3University Cancer Center Hamburg, University Medical Center, Hamburg, 4Department of Oncology and Hematology, University Medical Center, Hamburg This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies. Immunology and Infection Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus Christina Stoeckle1,2, Ioanna A. Rota1, Eva Tolosa3, Christoph Haller4, Arthur Melms5, Eleni Adamopoulou1 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.
