Eastern Virginia Medical School View Institution's Website 9 articles published in JoVE Neuroscience TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis Alisa A. Omelchenko1,2, Hua Bai1, Sibtain Hussain1, Jordan J. Tyrrell1,3, Mason Klein4, Lina Ni1 1School of Neuroscience, Virginia Polytechnic Institute and State University, 2CMU-Pitt Joint Computational Biology, School of Medicine, University of Pittsburgh, 3Eastern Virginia Medical School, 4Department of Physics, University of Miami TrackMate Analysis of Calcium Imaging (TACI) is an open-source ImageJ plugin for 3D calcium imaging analysis that examines motion on the z-axis and identifies the maximum value of each z-stack to represent a cell's intensity at the corresponding time point. It can separate neurons overlapping in the lateral (x/y) direction but on different z-planes. Biology CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis Charlotte Chambers1, Linh Quan1, Grace Yi1, Aurora Esquela-Kerscher1 1Department of Microbiology & Molecular Cell Biology, Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School This protocol describes a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) gene editing workflow for microRNA cluster network analysis that allows the rapid generation of a panel of genetically modified cell lines carrying unique miRNA cluster member deletion combinations as large as 35 kb within a single experiment. Biochemistry Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy David P. Klebl1, Frank Sobott2, Howard D. White3, Stephen P. Muench1 1School of Biomedical Sciences, Faculty of Biological Sciences & Astbury Centre for Structural and Molecular Biology, University of Leeds, 2School of Molecular and Cellular Biology, Faculty of Biological Sciences & Astbury Centre for Structural and Molecular Biology, University of Leeds, 3Department of Physiological Sciences, Eastern Virginia Medical School Here, we provide a detailed protocol for the use of a rapid grid making device for both fast grid-making and for rapid mixing and freezing to conduct time-resolved experiments. Immunology and Infection Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice Nagaraja Nagre1, Xiaofei Cong1, Andrew C. Pearson1, Xiaoli Zhao1 1Department of Physiological Sciences, Eastern Virginia Medical School Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice. Immunology and Infection Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue Bronson A. Haynes1, Ryan W. Huyck1, Ashley J. James1, Meghan E. Carter1, Omnia U. Gaafar1, Marjorie Day1, Avennette Pinto1, Anca D. Dobrian1 1Department of Physiological Sciences, Eastern Virginia Medical School The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed. Behavior Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach Frency Varghese1, Jessica A. Burket2, Andrew D. Benson2, Stephen I. Deutsch2, Christian W. Zemlin1 1Department of Electrical and Computer Engineering and Frank Reidy Center for Bioelectrics, Old Dominion University, 2Department of Psychiatry & Behavioral Sciences, Eastern Virginia Medical School This protocol describes a method to quantify mouse sociability. Mice are videotaped as they move and interact in a special cage. Movie processing allows for the automated quantification of sociability with excellent accuracy and reliability. Medicine A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys Liwei Huang1, An Xiao1, Andrea Wecker1, Daniel A. McBride1, Soo Young Choi2, Weibin Zhou3, Joshua H. Lipschutz2 1Department of Medicine, Eastern Virginia Medical School, 2Department of Medicine, Medical University of South Carolina, 3Department of Pediatrics, University of Michigan We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish. Bioengineering Bacterial Immobilization for Imaging by Atomic Force Microscopy David P. Allison1,2, Claretta J. Sullivan3, Ninell Pollas Mortensen1,2, Scott T. Retterer1,4, Mitchel Doktycz1,4 1Biological and Nanoscale Systems Group, Biosciences Division, Oak Ridge National Laboratory, 2Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, 3Department of Surgery, Eastern Virginia Medical School, 4Center for Nanophase Materials Sciences Division, Oak Ridge National Laboratory Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM). Immunology and Infection Flow Cytometry Analysis of Immune Cells Within Murine Aortas Matthew J. Butcher1, Margo Herre1, Klaus Ley2, Elena Galkina1 1Deptartment of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, 2Division of Inflammation Biology, LaJolla Institute for Allergy and Immunology This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.