Georgetown University Medical Center 9 articles published in JoVE 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. Genetics Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction Dan Zhou*1,2, Lei Tan*1, Jian Li*3, Tanbin Liu1, Yi Hu1, Yalan Li1, Sachiyo Kawamoto4, Chengyu Liu5, Shiyin Guo3, Aibing Wang1 1Lab of Animal Models and Functional Genomics (LAMFG), The Key Laboratory of Animal Vaccine & Protein Engineering, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), 2Department of Pathology, Georgetown University Medical School, 3College of Food Science and Technology, Hunan Agricultural University (HUNAU), 4Lab of Molecular Cardiology (LMC), National Heart, Lung, and Blood Institute (NHLBI)/National Institutes of Health (NIH), 5Transgenic Core, National Heart, Lung, and Blood Institute (NHLBI)/National Institutes of Health (NIH) Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology. 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. Biology Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy Parnika Kadam1,2, Ryan McAllister3, Jeffrey S. Urbach3, Kathryn Sandberg1,2, Susette C. Mueller4 1Department of Biochemistry, Georgetown University Medical Center, 2Department of Medicine, Georgetown University Medical Center, 3Department of Physics, Georgetown University Medical Center, 4Department of Oncology, Georgetown University Medical Center Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time. Cancer Research A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation Lucas Tricoli1, Deborah L. Berry2, Chris Albanese1 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. Cancer Research In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis Sung-Hyeok Hong*1, Jason U. Tilan*2,3, Susana Galli1, Rachel Acree3, Katherine Connors4, Akanksha Mahajan1, Larissa Wietlisbach3, Taylor Polk3, Ewa Izycka-Swieszewska5, Yi-Chien Lee6, Luciane R. Cavalli6, Olga C. Rodriguez6, Chris Albanese6,7, Joanna B. Kitlinska1 1Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, 2Department of Nursing, Georgetown University, School of Nursing and Health Studies, 3Department of Human Science, Georgetown University, School of Nursing and Health Studies, 4School of Medicine, Georgetown University Medical Center, 5Department of Pathology and Neuropathology, Medical University of Gdańsk, 6Department of Oncology, Georgetown University Medical Center, 7Department of Pathology, Georgetown University Medical Center This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types. Bioengineering Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis Stephanie M. Williams1, Lalitha Venkataraman1, Huilai Tian1, Galam Khan2, Brent T. Harris2,3, Michael R. Sierks1 1School for Engineering of Matter, Transport and Energy, Arizona State University, 2Department of Neurology, Georgetown University Medical Center, 3Department of Pathology, Georgetown University Medical Center Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis. Immunology and Infection Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates Stefano G. Daniele1, Amanda A. Edwards1, Kathleen A. Maguire-Zeiss1 1Department of Neuroscience, Georgetown University Medical Center One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam). Biology Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis Lyuba Khavrutskii1,2, Joanna Yeh1, Olga Timofeeva3, Sergey G. Tarasov4, Samuel Pritt1, Karen Stefanisko1, Nadya Tarasova1 1Cancer and Inflammation Program, National Cancer Institute, 2Basic Science Program, SAIC-Frederick, Inc., 3Departments of Oncology and Radiation Medicine, Georgetown University Medical Center, 4Structural Biophysics Laboratory, National Cancer Institute Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.