Georgetown University View Institution's Website 15 articles published in JoVE Immunology and Infection A Minimally Invasive, Accurate, and Efficient Technique for Intrathymic Injection in Mice Michael T. McGuire*1, Andrea Z. Tuckett*2, Faith Myint2, Johannes L. Zakrzewski2,3,4 1Department of Radiology, Hackensack University Medical Center, 2Center for Discovery and Innovation, Hackensack Meridian Health, 3Department of Pediatrics, Hackensack University Medical Center, 4Department of Oncology, Georgetown University The present protocol describes an interventional radiology procedure established for intrathymic injection in mice to avoid the risk of open surgery and improve the accuracy of blind percutaneous injections. Cancer Research Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures Ghada M. Sharif1, Leon Der2, Anna T. Riegel1, Makarand Paranjape2, Anton Wellstein1 1Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, 2Department of Physics, Georgetown University We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed. Medicine Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice Wenyu Gou*1, Wanxing Cui*2, Yuki Cui2, Hongjun Wang1,3 1Department of Surgery, Medical University of South Carolina, 2Georgetown University, 3Ralph H. Johnson Veterans Affairs Medical Center Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice. Cancer Research Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture Yuan-Na Lin1, Apsra Nasir1, Sharon Camacho1, Deborah L. Berry1, Marcel O. Schmidt1, Gray W. Pearson1, Anna T. Riegel1, Anton Wellstein1 1Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity. Genetics A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues Ranjan K. Behera1, Kevin D. Mlynek1, Matthew S. Linz1, Shaun R. Brinsmade1 1Department of Biology, Georgetown University Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection. Medicine A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury Bevan S. Main1, Stephanie S. Sloley1, Sonia Villapol1, David N. Zapple2, Mark P. Burns1 1Laboratory for Brain Injury and Dementia, Department of Neuroscience, Georgetown University Medical Center, 2University Information Systems, Division of Research Technologies, Georgetown University Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion. Biochemistry A Tandem Liquid Chromatography–Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus David J. Samuels1, Zhe Wang2, Kyu Y. Rhee2,3, Shaun R. Brinsmade1 1Department of Biology, Georgetown University, 2Division of Infectious Diseases, Weill Cornell Medical College, 3Department of Medicine, Weill Cornell Medical College Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry. Cancer Research Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio) Eric B. Berens1, Ghada M. Sharif1, Anton Wellstein1, Eric Glasgow1 1Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University This method utilizes zebrafish embryos to efficiently test the vascular invasive ability of cancer cells. Fluorescent cancer cells are injected into the precardiac sinus or yolk sac of developing embryos. Cancer cell vascular invasion and extravasation is assessed via fluorescence microscopy of the tail region 24 to 96 hr later. Neuroscience A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster Timothy P. Murphy1,2, Dan D. Luu1, Jessica A. DeSimone1,3, Thomas C. O'Brien1,4, Christopher J. Lally1,5, Jillian J. Lindblad1,6, Sarah M. Webster1 1Department of Biology, College of the Holy Cross, 2School of Medicine, Georgetown University, 3Department of Biochemistry, Giesel School of Medicine, Dartmouth College, 4School of Medicine, Tufts University, 5Transgenomic Inc., 6Department of Molecular, Cell and Cancer Biology, UMass Medical School In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal. Medicine A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting Eric B. Berens1, Jon M. Holy2, Anna T. Riegel1, Anton Wellstein1 1Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, 2Department of Biomedical Sciences, University of Minnesota This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored. Biology SpOT the Correct Tissue Every Time in Multi-tissue Blocks Anna Coffey1,2, Michael D. Johnson3, Deborah L. Berry3 1Center for Advanced Preclinical Research (CAPR), Frederick National Laboratory for Cancer Research, 2Leidos Biomedical Research, Inc., 3Department of Oncology, Georgetown University The purpose of the Specimen Orientation Tag (SpOT) is to function as an orientation tool to aid in individual tissue identification in multi-tissue paraffin blocks. These protocols demonstrate how it is constructed easily from common, low-cost histology materials and serves as a reliable visual marker in paraffin blocks and sections. Biology An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus Monica F. Poelchau1, Xin Huang1, Allison Goff1, Julie Reynolds2, Peter Armbruster1 1Department of Biology, Georgetown University, 2Insect Physiology Lab, EEOB, The Ohio State University RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data. Medicine Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer Rebecca E. Nakles1, Sarah L. Millman1, M. Carla Cabrera1, Peter Johnson1,2, Susette Mueller1,2, Philipp S. Hoppe3, Timm Schroeder3, Priscilla A. Furth1,2,4,5 1Department of Oncology, Georgetown University, 2Lombardi Comprehensive Cancer Center, Georgetown University, 3Stem Cell Dynamics, Helmholtz Zentrum München - German Research Center for Environmental Health, 4Department of Medicine, Georgetown University, 5Department of Nanobiomedical Science and WCU Research Center of Nanobiomedical Science, Dankook University Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions. Medicine A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells Said Rahim1, Aykut Üren1 1Lombardi Comprehensive Cancer Center, Georgetown University This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom. Biology Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats Sonia de Assis1, Anni Warri1,2, M. Idalia Cruz1, Leena Hilakivi-Clarke1 1Department of Oncology, Georgetown University, 2Institute of Biomedicine, University of Turku Medical Faculty Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.