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Eastern Virginia Medical School

12 ARTICLES PUBLISHED IN JoVE

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Biology

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems
Noah Weisleder 1, Peihui Lin 1, Xiaoli Zhao 1, Matthew Orange 1, Hua Zhu 1, Jianjie Ma 1
1Department of Physiology and Biophysics, Robert Wood Johnson Medical School

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

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Immunology and Infection

Flow Cytometry Analysis of Immune Cells Within Murine Aortas
Matthew J. Butcher 1, Margo Herre 1, Klaus Ley 2, Elena Galkina 1
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.

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Bioengineering

Bacterial Immobilization for Imaging by Atomic Force Microscopy
David P. Allison 1,2, Claretta J. Sullivan 3, Ninell Pollas Mortensen 1,2, Scott T. Retterer 1,4, Mitchel Doktycz 1,4
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).

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Biology

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber
Zui Pan 1, Xiaoli Zhao 2, Marco Brotto 3
1Department of Physiology and Biophysics, Confocal Microscopy and Cell Imaging Core, Robert Wood Johnson Medical School , 2Department of Physiology and Biophysics, Robert Wood Johnson Medical School , 3Muscle Biology Research Group-MUBIG Schools of Nursing & Medicine, University of Missouri-Kansas City

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

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Biology

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles
Ki Ho Park 1, Leticia Brotto 2, Oanh Lehoang 1, Marco Brotto 2, Jianjie Ma 1, Xiaoli Zhao 1,3
1Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, 2Muscle Biology Research Group, University of Missouri-Kansas City, 3Pharmacology division, College of Pharmacy, DHLRI, Ohio State University

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

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Medicine

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys
Liwei Huang 1, An Xiao 1, Andrea Wecker 1, Daniel A. McBride 1, Soo Young Choi 2, Weibin Zhou 3, Joshua H. Lipschutz 2
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.

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Behavior

Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach
Frency Varghese 1, Jessica A. Burket 2, Andrew D. Benson 2, Stephen I. Deutsch 2, Christian W. Zemlin 1
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.

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Immunology and Infection

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue
Bronson A. Haynes 1, Ryan W. Huyck 1, Ashley J. James 1, Meghan E. Carter 1, Omnia U. Gaafar 1, Marjorie Day 1, Avennette Pinto 1, Anca D. Dobrian 1
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.

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Immunology and Infection

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice
Nagaraja Nagre 1, Xiaofei Cong 1, Andrew C. Pearson 1, Xiaoli Zhao 1
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.

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Biochemistry

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy
David P. Klebl 1, Frank Sobott 2, Howard D. White 3, Stephen P. Muench 1
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.

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Biology

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis
Charlotte Chambers 1, Linh Quan 1, Grace Yi 1, Aurora Esquela-Kerscher 1
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.

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Neuroscience

TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis
Alisa A. Omelchenko 1,2, Hua Bai 1, Sibtain Hussain 1, Jordan J. Tyrrell 1,3, Mason Klein 4, Lina Ni 1
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.

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