University of Texas Southwestern View Institution's Website 11 articles published in JoVE Bioengineering Production of Membrane-Filtered Phase-Shift Decafluorobutane Nanodroplets from Preformed Microbubbles Darrah A. Merillat1, Arvin Honari1, Shashank R. Sirsi1,2 1Department of Bioengineering, The University of Texas at Dallas, 2Department of Radiology, The University of Texas Southwestern This protocol describes a method of generating large volumes of lipid encapsulated decafluorobutane microbubbles using probe-tip sonication and subsequently condensing them into phase-shift nanodroplets using high-pressure extrusion and mechanical filtration. Medicine A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells Ozlem Kulak1, Lawrence Lum1 1Department of Cell Biology, UT Southwestern Medical Center Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries. Bioengineering Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering Bahar Bilgen1,2, Danielle Chu3, Robert Stefani1, Roy K. Aaron1,2 1Department of Orthopaedics, The Warren Alpert Brown Medical School of Brown University and the Rhode Island Hospital, 2Center for Restorative and Regenerative Medicine, VA Medical Center, Providence, RI, 3University of Texas Southwestern Medical Center We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect. Biology Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets L. Evan Reddick1, Neal M. Alto1 1Department of Molecular Microbiology, University of Texas Southwestern Medical Center The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications. Immunology and Infection Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus Xiaonan Dong1, Pinghui Feng2 1Center for Autophagy Research, Department of Internal Medicine, UT Southwestern Medical Center, 2Department of Microbiology, UT Southwestern Medical Center We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (γHV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for γHV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication. Medicine In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model Debabrata Saha1, Henry Dunn2, Heling Zhou2, Hiroshi Harada3, Masahiro Hiraoka3, Ralph P. Mason2, Dawen Zhao2 1Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2Department of Radiology, University of Texas Southwestern Medical Center, 3Department of Radiation Oncology, Kyoto University Graduate School of Medicine Bioluminescence imaging of hypoxia inducible factor-1α activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model. Immunology and Infection RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract Eduardo Lopez-Medina1, Megan M. Neubauer1, Gerald B. Pier2, Andrew Y. Koh3 1Department of Pediatrics, University of Texas Southwestern Medical Center, 2Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 3Department of Pediatrics and Microbiology, University of Texas Southwestern Medical Center A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes. Neuroscience A Simple Way to Measure Ethanol Sensitivity in Flies Thomas Maples1, Adrian Rothenfluh1 1Department of Psychiatry, University of Texas Southwestern Medical Center A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described. Neuroscience The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice Kelly A. Tennant1, Aaron L. Asay2, Rachel P. Allred3, Angela R. Ozburn4, Jeffrey A. Kleim5, Theresa A. Jones1,2 1Institute for Neuroscience, University of Texas at Austin, 2Department of Psychology, University of Texas at Austin, 3Department of Neurology, University of Florida, 4Department of Psychiatry, University of Texas Southwestern Medical Center, 5Department of Neuroscience, McKnight Brain Institute, University of Florida The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments. Biology Method for the Isolation of Francisella tularensis Outer Membranes Jason F. Huntley1, Gregory T. Robertson1, Michael V. Norgard1 1Department of Microbiology, University of Texas Southwestern Medical Center A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation. Biology Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging W. Ryan Williamson1, P. Robin Hiesinger1 1Department of Physiology and Green Center for Systems Biology, University of Texas Southwestern Medical Center This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.