University of South Florida View Institution's Website 16 articles published in JoVE Medicine Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids Amelia Llerena1, Shaheda Urmi2, Jahanshah Amin3, Byeong Cha3, Thao TB Ho2 1Morsani College of Medicine, University of South Florida, 2Department of Pediatrics, Morsani College of Medicine, University of South Florida, 3Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida This protocol details the establishment of enteroids, a three-dimensional intestinal model, from fetal intestinal tissue. Immunofluorescent imaging of epithelial biomarkers was used for model characterization. Apical exposure of lipopolysaccharides, a bacterial endotoxin, using microinjection technique induced epithelial permeability in a dose-dependent manner measured by the leakage of fluorescent dextran. Biology A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage Yunhee Ji*1, Mahsa Karbaschi*2, Abdulhadi Abdulwahed*3, Natalia S. Quinete4, Mark D. Evans5, Marcus S. Cooke1 1Oxidative Stress Group, Dept. Cell Biology, Microbiology and Molecular Biology, College of Arts and Sciences, University of South Florida, 2Cepheid (Danaher Corp.), US Technical Operations, 3Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, 4Department of Chemistry and Biochemistry, Institute of Environment, Florida International University, 5Leicester School of Allied Health Sciences, Faculty of Health and Life Sciences, De Montfort University The comet assay is a popular means of detecting DNA damage. This study describes an approach to running slides in representative variants of the comet assay. This approach significantly increased the number of samples while decreasing assay run-time, the number of slide manipulations, and the risk of damage to gels. Biochemistry Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions Robert P. Sparks*1, William Lawless*2, Andres S. Arango*1,3,4, Emad Tajkhorshid1,3,4, Rutilio A. Fratti1,3 1Department of Biochemistry, University of Illinois at Urbana-Champaign, 2Department of Chemistry, University of South Florida, 3Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, 4Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements. Immunology and Infection Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model Alexa D. Gannon1, Sophie E. Darch1 1Department of Molecular Medicine, Morsani College of Medicine, University of South Florida Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies. Cancer Research A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease Vincent Law1,2, Margi Baldwin3, Ganesan Ramamoorthi4, Krithika Kodumudi4, Nam Tran1, Inna Smalley5, Derek Duckett6, Pawel Kalinski7, Brian Czerniecki4, Keiran S. M. Smalley2, Peter A. Forsyth1,2 1Department of Neuro-Oncology, H. Lee Moffitt Cancer Center & Research Institute, 2Department of Tumor Biology, H. Lee Moffitt Cancer Center & Research Institute, 3Department of Comparative Medicine, University of South Florida, 4Department of Breast Oncology, H. Lee Moffitt Cancer Center & Research Institute, 5Department of Cancer Physiology, H. Lee Moffitt Cancer Center & Research Institute, 6Department of Drug Discovery, H. Lee Moffitt Cancer Center & Research Institute, 7Department of Medical Oncology, Roswell Park Comprehensive Cancer Center Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease. Immunology and Infection Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria Robert S. Brzozowski1, Maria L. White1, Prahathees J. Eswara1 1Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus. Engineering Method Development for Contactless Resonant Cavity Dielectric Spectroscopic Studies of Cellulosic Paper Mary Kombolias1, Jan Obrzut2, Michael T. Postek3,4, Dianne L. Poster2, Yaw S. Obeng3 1Testing and Technical Services, Plant Operations, United States Government Publishing Office, 2Materials Measurement Laboratory, National Institute of Standards and Technology, 3Nanoscale Device Characterization Division, Physical Measurement Laboratory, National Institute of Standards and Technology, 4College of Pharmacy, University of South Florida A protocol for the non-destructive analysis of the fiber content and relative age of paper. Medicine Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques Elizabeth M. Johnson1, Kenneth L. Gage2, Sebastian Feuerlein2, Daniel Jeong2 1Department of Radiology, University of South Florida, 2Department of Diagnostic Imaging, H. Lee Moffitt Cancer Center and Research Institute The goal of this article is to describe how cardiac magnetic resonance can be used for the evaluation and diagnosis of a suspected cardiac thrombus. The method presented will describe data acquisition as well as the pre-procedure and post-procedure protocol. Behavior Using a Real-Time Locating System to Measure Walking Activity Associated with Wandering Behaviors Among Institutionalized Older Adults Mary E. Bowen1,2, William Kearns3, Jeremy R. Crenshaw4, Steven J. Stanhope4 1School of Nursing, University of Delaware, 2Corporeal Michael J. Crescenz VA Medical Center, 3Child and Family Studies, University of South Florida, 4Department of Kinesiology and Applied Physiology, University of Delaware This paper discusses the use of a continuous and objective real-time locating system to measure walking activity associated with wandering behaviors, focusing on older adults with cognitive impairment. Walking activity is measured by walking distance, sustained walking distance, and sustained gait speed. Also assessed are gait quality and balance ability. Behavior Elevated Plus Maze Test Combined with Video Tracking Software to Investigate the Anxiolytic Effect of Exogenous Ketogenic Supplements Csilla Ari1, Dominic P. D’Agostino2,3, David M. Diamond1,2, Mark Kindy4,5,6, Collin Park1, Zsolt Kovács7 1Department of Psychology, Hyperbaric Neuroscience Research Laboratory, University of South Florida, 2Department of Molecular Pharmacology and Physiology, Metabolic Medicine Research Laboratory, Morsani College of Medicine, University of South Florida, 3Institute for Human and Machine Cognition, 4Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, 5James A. Haley VA Medical Center, 6Shriners Hospital for Children, 7Savaria Department of Biology, ELTE Eötvös Loránd University Here, we present a protocol to investigate changes in the anxiety level of rodent animal models. The elevated plus maze (EPM) test, used together with a video tracking software, provides a reliable method to document the effect of various potential anxiolytic treatments in preclinical laboratory scenarios. Engineering Determining Tribocorrosion Rate and Wear-Corrosion Synergy of Bulk and Thin Film Aluminum Alloys Jia Chen1,2, Hesham Mraied3, Wenjun Cai1,2 1Department of Mechanical Engineering, University of South Florida, 2Department of Materials Science and Engineering, Virginia Polytechnic Institute and State University, 3Department of Civil and Environmental Engineering, University of South Florida Here, we present a protocol to measure the tribocorrosion rate and wear-corrosion synergy of thin film and bulk Al alloys in simulated sea water at room temperature. Environment Using Pharmacological Manipulation and High-precision Radio Telemetry to Study the Spatial Cognition in Free-ranging Animals Timothy C. Roth*1, Aaron R. Krochmal*2, William B. Gerwig, IV1, Sage Rush3, Nathaniel T. Simmons2, Jeffery D. Sullivan4, Katrina Wachter5 1Department of Psychology, Franklin and Marshall College, 2Department of Biology, Washington College, 3University of Pennsylvania, 4School of Forestry and Wildlife Sciences, Auburn University, 5Morsani College of Medicine, University of South Florida This paper describes a novel protocol that combines the pharmacological manipulation of memory and radio telemetry to document and quantify the role of cognition in navigation. Chemistry Sediment Core Extrusion Method at Millimeter Resolution Using a Calibrated, Threaded-rod Patrick T. Schwing1, Isabel C. Romero1, Rebekka A. Larson2, Bryan J. O'Malley1, Erika E. Fridrik1, Ethan A. Goddard1, Gregg R. Brooks2, David W. Hastings2, Brad E. Rosenheim1, David J. Hollander1, Guy Grant1, Jim Mulhollan1 1College of Marine Science, University of South Florida, 2Marine Science and Chemistry, Eckerd College An extrusion method using a calibrated threaded-rod is presented, which allows for mm scale subsampling of aquatic sediment cores. Millimeter-scale sampling is necessary to fully characterize recent event stratigraphy in sediment records. Medicine Demonstration of the Rat Ischemic Skin Wound Model Andrea N. Trujillo*1, Shannon L. Kesl*1,2, Jacob Sherwood1, Mack Wu2, Lisa J. Gould1,3 1Department of Molecular Pharmacology and Physiology, University of South Florida, 2Department of Surgery, University of South Florida, 3Wound Recovery and Hyperbaric Medicine Center, Kent Memorial Hospital The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds. Biology Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome Lancia N.F. Darville1, Bernd H.A. Sokolowski1 1Department of Otolaryngology, Morsani College of Medicine, University of South Florida Proteome analysis of the cochlear sensory epithelium can be challenging due to its small size and because membrane proteins are difficult to isolate and identify. Both membrane and soluble proteins can be identified by combining multiple preparative methods and separation techniques along with high-resolution mass spectrometry. Medicine Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS) Nadine Nelson1, Karoly Szekeres1, Denise Cooper1, Tomar Ghansah1 1Department of Molecular Medicine, University of South Florida Morsani College of Medicine This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.
