Medical University of Graz View Institution's Website 17 articles published in JoVE Developmental Biology Ex Vivo Placental Explant Flow Culture - Mimicking the Dynamic Conditions In Utero Nadja Kupper1, Elisabeth Pritz1, Monika Siwetz1, Jacqueline Guettler1, Berthold Huppertz1 1Division of Cell Biology, Histology and Embryology, Gottfried Schatz Research Center, Medical University of Graz Here is a protocol for culturing placental explants under constant flow conditions. This approach enhances traditional static villous culture systems by enabling the replication of dynamic physiological environments. Cancer Research Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells Tommaso Sconocchia1, Johannes Foßelteder1, Thomas Köhnke2, Ravindra Majeti2, Andreas Reinisch1,3 1Division of Hematology, Department of Internal Medicine, Medical University of Graz, 2Division of Hematology, Stanford Cancer Institute, Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 3Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described. Cancer Research Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy Hansjörg Habisch*1, Fangrong Zhang*1,2, Qishun Zhou1, Tobias Madl1 1Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Molecular Biology and Biochemistry, Medical University of Graz, 2Ministry of Education, School of Basic Medical Sciences, Key Laboratory of Gastrointestinal Cancer (Fujian Medical University) The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy. Neuroscience Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines Muammer Üçal*1, Michaela Tanja Haindl*2, Milena Z. Adzemovic3, Manuel Zeitelhofer4, Ute Schaefer1, Franz Fazekas2, Sonja Hochmeister2 1Research Unit of Experimental Neurotraumatology, Department of Neurosurgery, Medical University Graz, 2Department of Neurology, Medical University Graz, 3Centre for Molecular Medicine, Department of Clinical Neuroscience, Karolinska Institutet, 4Division of Vascular Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter. Medicine International Expert Consensus and Recommendations for Neonatal Pneumothorax Ultrasound Diagnosis and Ultrasound-guided Thoracentesis Procedure Jing Liu1,2, Dalibor Kurepa3, Francesco Feletti4,5, Almudena Alonso-Ojembarrena6, Jovan Lovrenski7, Roberto Copetti8, Erich Sorantin9, Javier Rodriguez-Fanjul10, Karishma Katti3, Andrea Aliverti4, Huayan Zhang11,12, Misun Hwang13, Tsu F. Yeh14, Cai-Bao Hu15, Xing Feng16, Ru-Xin Qiu1,2, Jing-Han Chi17, Li-Li Shang18, Guo-Rong Lyu19, Shao-Zheng He20, Yan-Fen Chai21, Zhan-Jun Qiu22, Hai-Ying Cao2,23, Yue-Qiao Gao1,2, Xiao-Ling Ren1,2, Guo Guo1,24, Li Zhang1,2, Ying Liu1,2, Wei Fu1,2, Zu-Lin Lu1,2, Hong-Lei Li1,2 1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The National Neonatal Lung Ultrasound Training Base, 3 Pneumothorax is a common emergency and critical disease in newborn infants that needs rapid, clear diagnosis and timely treatment. Diagnosis and treatment based on chest X-rays are associated with delayed management and radiation damage. Lung ultrasound (US) provides useful guidance for rapid, accurate diagnosis and the precise thoracentesis of pneumothorax. Medicine Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse Bakytbek Egemnazarov1, Grazyna Kwapiszewska1,2, Leigh M. Marsh1 1Ludwig Boltzmann Institute for Lung Vascular Research, 2Department of Physiology, Otto Loewi Research Center, Medical University of Graz Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably. Neuroscience An Unbiased Approach of Sampling TEM Sections in Neuroscience Stefan Wernitznig1, Florian Reichmann2, Mariella Sele1, Christoph Birkl3, Johannes Haybäck4,5, Florian Kleinegger4, Anna Birkl-Töglhofer4, Stefanie Krassnig4, Christina Wodlej4, Peter Holzer2, Daniel Kummer1, Elisabeth Bock1, Gerd Leitinger1 1Department of Cell Biology, Histology and Embryology, Gottfried Schatz Research Center, Medical University of Graz, 2Department of Pharmacology, Otto Loewi Research Center, Medical University of Graz, 3Division of General Neurology, Department of Neurology, Medical University of Graz, 4Institute of Pathology, Medical University of Graz, 5Department of Pathology, Medical Faculty, Otto von Guericke University Magdeburg We introduce a novel workflow for electron microscopy investigations of brain tissue. The method allows the user to examine neuronal features in an unbiased fashion. For elemental analysis, we also present a script that automatizes most of the workflow for randomized sampling. Medicine Protocol and Guidelines for Point-of-Care Lung Ultrasound in Diagnosing Neonatal Pulmonary Diseases Based on International Expert Consensus Jing Liu1,2, Roberto Copetti3, Erich Sorantin4, Jovan Lovrenski5, Javier Rodriguez-Fanjul6, Dalibor Kurepa7, Xing Feng8, Luigi Cattaross9, Huayan Zhang10,11, Misun Hwang12, Tsu F. Yeh13,14, Yisrael Lipener7, Abhay Lodha15, Jia-Qin Wang16, Hai-Ying Cao2,17, Cai-Bao Hu2,18, Guo-Rong Lyu19, Xin-Ru Qiu1,2, Li-Qun Jia20, Xiao-Man Wang20, Xiao-Ling Ren1,2, Jiu-Ye Guo1,2, Yue-Qiao Gao1,2, Jian-Jun Li1,2, Ying Liu1,2, Wei Fu1,2, Yan Wang21, Zu-Lin Lu1,2, Hua-Wei Wang8, Li-Li Shang22 1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The Neonatal Lung Ultrasound Training Base, Chinese College of Critical Ultrasound, 3Emergency Department, Cattinara University Hospital, 4Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 5Faculty of Medicine, University of Novi Sad, Radiology Department, Institute for Children and Adolescents Health Care of Vojvodina, 6Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 7Department of Neonatology, Children's Hospital of Soochow University, 9Department of Neonatology, Udine University Hospital, 10Division of Neonatology, Children's Hospital of Philadelphia, 12Intensive Care Unit, Zhejiang Hospital, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Neonatology and NICU, Tai'an City Central Hospital of Shandong Province, 22Department of Intensive Care Unit, The Second Affiliated Hospital of Heilongjiang University of Chinese Medicine Lung ultrasound is a noninvasive and valuable tool for bedside evaluation of neonatal lung diseases. However, a relative lack of reference standards, protocols and guidelines may limit its application. Here, we aim to develop a standardized neonatal lung ultrasound diagnostic protocol to be used in clinical decision-making. 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). Medicine Oral Biofilm Sampling for Microbiome Analysis in Healthy Children Elisabeth Santigli1, Martin Koller2, Barbara Klug1 1Division of Oral Surgery and Orthodontics, Department of Dental Medicine and Oral Health, Medical University of Graz, 2Division of Preventive and Operative Dentistry, Periodontology, Prosthodontics and Restorative Dentistry, Department of Dental Medicine and Oral Health, Medical University of Graz Changes in the oral microbiome throughout childhood are of growing interest. Comparison of different microbiome studies reveals a lack of standardized sampling protocols. Limited space makes sampling the sound subgingival sulcus of children challenging. Paper point sampling is presented here in detail as the method of choice for this area. Cancer Research Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation Martina Loibner1,2, Lisa Oberauner-Wappis1,2, Christian Viertler2, Daniel Groelz3, Kurt Zatloukal1,2 1Christian Doppler Laboratory for Biospecimen Research and Biobanking Technologies, Institute of Pathology, Medical University Graz, 2Institute of Pathology, Medical University Graz, 3Research and Development, Qiagen GmbH Fluorescence in-situ hybridization (FISH) is often required in combination with histopathology and molecular diagnostics for selection of therapy in personalized medicine. A novel non-cross-linking, formalin-free tissue fixative that allows high quality morphologic, molecular and FISH analyses from the same specimen by addition of a post-fixation step before FISH is presented. Developmental Biology Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies Roman Crazzolara1, Gabriele Kropshofer1, Michael Steurer2, Sieghart Sopper2,3, Wolfgang Schwinger4 1Department of Pediatrics, Medical University Innsbruck, 2Department of Internal Medicine V (Hematology & Oncology), Medical University Innsbruck, 3Tyrolean Cancer Research Institute, 4Department of Pediatrics, Medical University Graz Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment. Biology Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells Emrah Eroglu1, Rene Rost1, Helmut Bischof1, Sandra Blass1, Anna Schreilechner1, Benjamin Gottschalk1, Maria R. Depaoli1, Christiane Klec1, Suphachai Charoensin1, Corina T. Madreiter-Sokolowski1, Jeta Ramadani1, Markus Waldeck-Weiermair1, Wolfgang F. Graier1, Roland Malli1 1Institute of Molecular Biology and Biochemistry, Medical University of Graz This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO•) probes (geNOps) to monitor single cell NO• fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO• formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor. Medicine Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy Barbara Klug1,2, Claudia Rodler1, Martin Koller3, Gernot Wimmer3, Harald H. Kessler2, Martin Grube4, Elisabeth Santigli1 1Department of Orthodontics and Maxillofacial Orthopedics, Medical University of Graz, 2Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 3Department of Prosthodontics, Restorative Dentistry, Periodontology and Implantology, Medical University of Graz, 4Institute of Plant Sciences, Karl-Franzens-University Graz We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing. Biology Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures Katharina Schallmoser1, Dirk Strunk1 1Stem Cell Research Unit, Medical University of Graz, Austria Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments. Biology Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells Nicole A. Hofmann1, Andreas Reinisch1, Dirk Strunk1 1Stem Cell Research Unit, Medical University of Graz, Austria Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation. Biology Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs) Andreas Reinisch1, Dirk Strunk1 1Stem Cell Research Unit, Medical University of Graz, Austria This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.