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Georgetown University

17 ARTICLES PUBLISHED IN JoVE

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Biology

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats
Sonia de Assis 1, Anni Warri 1,2, M. Idalia Cruz 1, Leena Hilakivi-Clarke 1
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.

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Medicine

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells
Said Rahim 1, Aykut Üren 1
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.

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Medicine

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer
Rebecca E. Nakles 1, Sarah L. Millman 1, M. Carla Cabrera 1, Peter Johnson 1,2, Susette Mueller 1,2, Philipp S. Hoppe 3, Timm Schroeder 3, Priscilla A. Furth 1,2,4,5
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.

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Biology

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus
Monica F. Poelchau 1, Xin Huang 1, Allison Goff 1, Julie Reynolds 2, Peter Armbruster 1
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.

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Biology

SpOT the Correct Tissue Every Time in Multi-tissue Blocks
Anna Coffey 1,2, Michael D. Johnson 3, Deborah L. Berry 3
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.

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Medicine

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting
Eric B. Berens 1, Jon M. Holy 2, Anna T. Riegel 1, Anton Wellstein 1
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.

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Neuroscience

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster
Timothy P. Murphy 1,2, Dan D. Luu 1, Jessica A. DeSimone 1,3, Thomas C. O'Brien 1,4, Christopher J. Lally 1,5, Jillian J. Lindblad 1,6, Sarah M. Webster 1
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.

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Cancer Research

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)
Eric B. Berens 1, Ghada M. Sharif 1, Anton Wellstein 1, Eric Glasgow 1
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.

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Cancer Research

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation
Lucas Tricoli 1, Deborah L. Berry 2, Chris Albanese 1
1Department of Oncology, Lombardi Comprehensive Cancer Center, 2Department of Oncology, Georgetown University Medical Center

Here, we present a method for the establishment of a rapid in vitro system that supports the three dimensional culturing and subsequent luminal differentiation of primary prostate epithelial cells.

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Biochemistry

A Tandem Liquid Chromatography–Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus
David J. Samuels 1, Zhe Wang 2, Kyu Y. Rhee 2,3, Shaun R. Brinsmade 1
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.

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Medicine

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury
Bevan S. Main 1, Stephanie S. Sloley 1, Sonia Villapol 1, David N. Zapple 2, Mark P. Burns 1
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.

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Biology

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish
Steven Cassar 1, Xin Huang 2, Todd Cole 3
1Preclinical Safety, AbbVie, 2Statistics, AbbVie, 3Integrated Science and Technology, AbbVie

The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

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Genetics

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues
Ranjan K. Behera 1, Kevin D. Mlynek 1, Matthew S. Linz 1, Shaun R. Brinsmade 1
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.

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Cancer Research

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture
Yuan-Na Lin 1, Apsra Nasir 1, Sharon Camacho 1, Deborah L. Berry 1, Marcel O. Schmidt 1, Gray W. Pearson 1, Anna T. Riegel 1, Anton Wellstein 1
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.

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Medicine

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice
Wenyu Gou *1, Wanxing Cui *2, Yuki Cui 2, Hongjun Wang 1,3
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.

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Cancer Research

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures
Ghada M. Sharif 1, Leon Der 2, Anna T. Riegel 1, Makarand Paranjape 2, Anton Wellstein 1
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.

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Immunology and Infection

A Minimally Invasive, Accurate, and Efficient Technique for Intrathymic Injection in Mice
Michael T. McGuire *1, Andrea Z. Tuckett *2, Faith Myint 2, Johannes L. Zakrzewski 2,3,4
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.

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