Erasmus University Medical Center View Institution's Website 15 articles published in JoVE Bioengineering The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies Bram Meijlink1, Ilya Skachkov1, Antonius F. W. van der Steen1,2, Nico de Jong1,2, Klazina Kooiman1 1Department of Biomedical Engineering, Thoraxcenter, Erasmus MC University Medical Center Rotterdam, 2Laboratory of Acoustical Wavefield Imaging, Faculty of Applied Sciences, Delft University of Technology This protocol describes three methods on how to obtain and use 5 to 8-day old chicken embryos and their chorioallantoic membrane (CAM) as an in vivo model to study contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. Biochemistry Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry Karel Bezstarosti1, Lennart van der Wal1, Jeroen A. A. Demmers1 1Proteomics Center, Erasmus University Medical Center We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis. Genetics Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example Bas B. Oude Munnink1, David F. Nieuwenhuijse1, Reina S. Sikkema1, Marion Koopmans1 1ErasmusMC, Department of Viroscience, WHO Collaborating Centre for Arbovirus and Viral Hemorrhagic Fever Reference and Research, Erasmus University Medical Center We previously validated a protocol for amplicon-based whole genome Usutu virus (USUV) sequencing on a nanopore sequencing platform. Here, we describe the methods used in more detail and determine the error rate of the nanopore R10 flow cell. Genetics Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR Antonio Mancarella1, Francesco A. Procopio1, Tilmann Achsel2, Elisa De Crignis3, Brian T. Foley4, Giampietro Corradin5, Claudia Bagni2, Giuseppe Pantaleo1, Cecilia Graziosi1 1Division of Immunology and Allergy, Lausanne University Hospital, 2Department of Fundamental Neuroscience, University of Lausanne, 3Department of Biochemistry, Erasmus Medical Center, 4Theoretical Biology and Biophysics Group, Los Alamos National Laboratories, 5Department of Biochemistry, University of Lausanne RNA hairpins and loops can function as primers for reverse transcription (RT) in absence of sequence-specific primers, interfering with the study of overlapping antisense transcripts. We have developed a technique able to identify strand-specific RNA, and we have used it to study HIV-1 antisense protein ASP. 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. Cancer Research Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia: From Patient Material To Sequence Interpretation Andreas Agathangelidis*1, Lesley Ann Sutton*2,3, Anastasia Hadzidimitriou1, Cristina Tresoldi4, Anton W. Langerak5, Chrysoula Belessi6, Frederic Davi7, Richard Rosenquist2,3, Kostas Stamatopoulos1,2, Paolo Ghia8 1Institute of Applied Biosciences, Centre for Research and Technology Hellas, 2Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, 3Department of Molecular Medicine and Surgery, Karolinska Institutet, 4Division of Immunology, Transplantation and Infectious, IRCCS San Raffaele Scientific Institute, 5Department of Immunology, Laboratory for Medical Immunology, Erasmus University Medical Center, 6Hematology Department, Nikea General Hospital, 7Assistance publique - Hôpitaux de Paris (AP-HP), Hopital Pitié-Salpêtrière, Department of Hematology, and UPMC University Paris 06, UMRS 1138, 8Division of Experimental Oncology, IRCCS Istituto Scientifico San Raffaele and Università Vita-Salute San Raffaele Herein, we present a protocol that details the technical aspects and essential requirements to ensure robust IG gene sequence analysis in patients with chronic lymphocytic leukemia (CLL), based on the accumulated experience of the European Research initiative on CLL (ERIC). Medicine Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model Panithi Sukho1,2,3, Geesien S.A. Boersema4, Nicole Kops5, Johan F. Lange4, Jolle Kirpensteijn1,6, Jan Willem Hesselink1, Yvonne M. Bastiaansen-Jenniskens5, Femke Verseijden1,5 1Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, 2Department of Otorhinolaryngology, Erasmus MC University Medical Center, 3Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, 4Department of Surgery, Erasmus MC University Medical Center, 5Department of Orthopaedics, Erasmus MC University Medical Center, 6 The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage. Bioengineering The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells Matthias Schewe1, Andrea Sacchetti1, Mark Schmitt1, Riccardo Fodde1 1Department of Pathology, Erasmus MC Cancer Institute, Erasmus University Medical Center Intestinal organoid cultures are established from whole crypts and do not allow the analysis of self-renewal and differentiation in a cell-specific fashion. This protocol describes reconstitution of sorted stem (Lgr5+) and niche (Paneth) cells, which give rise to organoids while enabling their prior biochemical and genetic modification and functional analysis. Cancer Research Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples Simone Punt1, Robert J. Baatenburg de Jong2, Ekaterina S. Jordanova1,3 1Department of Pathology, Leiden University Medical Center, 2Department of Otorhinolaryngology and Head and Neck Surgery, Erasmus University Medical Center, 3Center for Gynecological Oncology Amsterdam, VU Medical Center Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer. Developmental Biology Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection Jean-Marie Delalande1,2, Nikhil Thapar1, Alan J. Burns1,3 1Birth Defects Research Centre, UCL Institute of Child Health, 2Blizard Institute, Centre for Digestive Diseases, Queen Mary University of London, Barts and The London School of Medicine and Dentistry, 3Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam Here we report dual labeling of neural crest cells and blood vessels using chickGFP neural tube intraspecies grafting combined with intra-vascular DiI injection. This experimental technique allows us to simultaneously visualize and study development of the NCC-derived (enteric) nervous system and the vascular system, during organogenesis. Biology Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells Tahsin Stefan Barakat1, Joost Gribnau1 1Department of Reproduction and Development, Erasmus MC - University Medical Center Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells. Neuroscience Three Dimensional Vestibular Ocular Reflex Testing Using a Six Degrees of Freedom Motion Platform Joyce Dits1, Mark M.J. Houben2, Johannes van der Steen1 1Department of Neuroscience, Erasmus MC, 2TNO Human Factors A method is described to measure three-dimensional vestibulo ocular reflexes (3D VOR) in humans using a six degrees of freedom (6DF) motion simulator. The gain and misalignment of the 3D angular VOR provide a direct measure of the quality of vestibular function. Representative data on healthy subjects are provided Neuroscience Video-oculography in Mice Marcel de Jeu1, Chris I. De Zeeuw1,2 1Department of Neuroscience, Erasmus MC, Rotterdam, The Netherlands, 2Department of Neuroscience, Royal Dutch Academy of Arts & Sciences (KNAW) Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors. Immunology and Infection One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures Wendy L.J. Hansen1, Judith Beuving1, Annelies Verbon2, Petra. F.G. Wolffs1 1Department of Medical Microbiology, Maastricht University Medical Center, 2Department of Internal Medicine, Erasmus Medical Center The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy. Biology Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM Kelly Morgan1, Michael Kharas1, Elaine Dzierzak2, D. Gary Gilliland1,3 1Department of Hematology and Oncology, Brigham and Women's Hospital and Harvard Medical School, 2Department of Cell Biology and Genetics, Erasmus University Medical Center, 3Department of Medicine, Howard Hughes Medical Institute, Brigham and Women's Hospital and Harvard Medical School This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.