Heidelberg University Hospital 14 articles published in JoVE Immunology and Infection Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 (BSL-3) Conditions Megan L. Stanifer1,2, Steeve Boulant2,3,4 1Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, 2Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, 3Department of Infectious Diseases, Virology, Heidelberg University Hospital, 4Research Group “Cellular Polarity and Viral Infection”, German Cancer Research Center (DKFZ) This protocol describes how to infect human intestinal organoids from either their apical or basolateral side to characterize host/pathogen interactions at the single-cell level using single-cell RNA sequencing (scRNAseq) technology. Cancer Research Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq Ashwin Narayanan1, Enrique Blanco-Carmona2, Engin Demirdizen1, Xueyuan Sun1,3, Christel Herold-Mende4, Matthias Schlesner2, Sevin Turcan1 1Neurology Clinic and National Center for Tumor Diseases, University Hospital Heidelberg, 2Bioinformatics and Omics Data Analytics, German Cancer Research Center (DKFZ), 3Clinical Cooperation Unit Neurooncology, German Cancer Research Center (DKFZ), 4Division of Experimental Neurosurgery, Department of Neurosurgery, University of Heidelberg, INF 400, Heidelberg, Germany Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. We developed a simple and efficient protocol that utilizes a combination of buffers and gradient centrifugation to isolate single nuclei from fresh frozen glioma tissues for single nucleus RNA and ATAC sequencing studies. Medicine Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model Patrick Haubruck1,2, Aimee C. Colbath1,3, Yolanda Liu1, Shihani Stoner1, Cindy Shu1, Christopher B. Little1 1Raymond Purves Bone and Joint Research Laboratory, Institute of Bone and Joint Research, Kolling Institute, Royal North Shore Hospital, University of Sydney, 2HTRG - Heidelberg Trauma Research Group, Center for Orthopedics, Trauma Surgery and Spinal Cord Injury, Trauma and Reconstructive Surgery, Heidelberg University Hospital, 3Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis. Developmental Biology Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils Soah Lee*1,2,3, Huaxiao Yang*1,2,3, Caressa Chen*1,2,3, Sneha Venkatraman1,2,3, Adrija Darsha1,2,3, Sean M. Wu1,2,3, Joseph C. Wu1,2,3, Timon Seeger1,2,3,4,5 1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Department of Medicine, Division of Cardiovascular Medicine, Stanford University, 3Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, 4Department of Medicine III, University Hospital Heidelberg, 5German Centre for Cardiovascular Research (DZHK), Partner Site Heidelberg/Mannheim This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes. Cancer Research Cell Cycle-specific Measurement of γH2AX and Apoptosis After Genotoxic Stress by Flow Cytometry Ramon Lopez Perez1,2, Franziska Münz1,2, Jonas Kroschke1,2, Jannek Brauer1,2, Nils H. Nicolay1,2,3, Peter E. Huber1,2 1CCU Molecular and Radiation Oncology, German Cancer Research Center, 2Department of Radiation Oncology, Heidelberg University Hospital, 3Department of Radiation Oncology, Freiburg University Medical Center The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure. Immunology and Infection Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection Thomas Tu1,2, Stephan Urban1,3 1Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, 2Gastroenterology and Liver Research Laboratory, Ingham Institute, 3German Center for Infection Research (DZIF) We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1–2 copies) integration using inverse nested PCR. Developmental Biology CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer Weijun Feng*1, Lena Herbst*2, Peter Lichter2, Stefan M. Pfister3,4,5, Hai-Kun Liu1, Daisuke Kawauchi3,4 1Division of Molecular Neurogenetics, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, 2Division of Molecular Genetics, German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), 3 Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum. Engineering Protocol for the Evaluation of MRI Artifacts Caused by Metal Implants to Assess the Suitability of Implants and the Vulnerability of Pulse Sequences Tim Hilgenfeld1, Marcel Prager1, Franz S. Schwindling2, Johann M.E. Jende1, Peter Rammelsberg2, Martin Bendszus1, Sabine Heiland1, Alexander Juerchott1 1Department of Neuroradiology, Heidelberg University Hospital, 2Department of Prosthodontics, Heidelberg University Hospital We describe a standardized method to evaluate magnetic resonance imaging artifacts caused by implants to estimate the suitability of the implants for magnetic resonance imaging and/or the vulnerability of different pulse sequences to metallic artifacts simultaneously. Developmental Biology Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes Irene U. Wacker1,2, Lisa Veith3, Waldemar Spomer2,4, Andreas Hofmann2,4, Marlene Thaler5, Stefan Hillmer6, Ulrich Gengenbach2,4, Rasmus R. Schröder1,2,3 1Cryo Electron Microscopy, Centre for Advanced Materials, Universität Heidelberg, 2Heidelberg Karlsruhe Research Partnership (HEiKA), 3Cryo Electron Microscopy, BioQuant, Universitätsklinikum Heidelberg, 4Institute for Automation and Applied Computer Science, Karlsruhe Institute of Technology (KIT), 5Carl Zeiss Microscopy GmbH, 6Electron Microscopy Core Facility, Universität Heidelberg This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM. Neuroscience Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry Asa S. Hidmark1, Peter P. Nawroth1,2,3, Thomas Fleming1,3 1Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg, 2Institute for Diabetes and Cancer IDC Helmholtz Center Munich, Germany & Joint Heidelberg-IDC Translational Diabetes Program, 3German Center for Diabetes Research (DZD) Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice. Medicine In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging Angelika Hoffmann1,2, Xavier Helluy2,3, Manuel Fischer2, Ann-Kristin Mueller4,5, Sabine Heiland2, Mirko Pham1,6, Martin Bendszus1, Johannes Pfeil4,5,7 1Department of Neuroradiology, Heidelberg University Hospital, 2Division of Experimental Radiology, Department of Neuroradiology, Heidelberg University Hospital, 3NeuroImaging Centre Research, Department of Neuroscience, Ruhr-University Bochum, 4Centre for Infectious Diseases, Parasitology Unit, Heidelberg University Hospital, 5German Centre for Infection Research (DZIF), 6Department of Neuroradiology, University of Würzburg, 7Center for Childhood and Adolescent Medicine, General Pediatrics, Heidelberg University Hospital We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging. Medicine Subcutaneous Trigeminal Nerve Field Stimulation for Refractory Facial Pain Martin Jakobs1, Sigrid Schuh-Hofer2, Andreas Unterberg1, Rezvan Ahmadi1 1Department of Neurosurgery, University Hospital Heidelberg, 2Center for Biomedicine and Medical Technology, University Hospital Mannheim Treating chronic or neuropathic facial pain can be challenging when medical or standard treatment fails. Subcutaneous nerve field stimulation is the least invasive form of neuromodulation and is used for chronic back pain. We applied this technology to treat chronic and neuropathic trigeminal facial pain. Medicine A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts Shaoping Hou1, Armin Blesch2, Paul Lu3,4 1Spinal Cord Research Center, Department of Neurobiology & Anatomy, Drexel University College of Medicine, 2Spinal Cord Injury Center, Heidelberg University Hospital, 3Veterans Administration Medical Center, San Diego, CA, 4Department of Neurosciences, University of California, San Diego We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats. Immunology and Infection An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei Ann-Kristin Mueller1, Jochen Behrends2, Jannike Blank2, Ulrich E. Schaible2, Bianca E. Schneider2 1Department of Infectious Diseases, Parasitology Unit, University Hospital Heidelberg, 2Priority Area Infections, Research Center Borstel Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.