University of Miami Miller School of Medicine 19 articles published in JoVE Cancer Research Preparation of Cytoplasmic and Nuclear Long RNAs from Primary and Cultured Cells Jacob Jahn1, Sana Chaudhry1, Maurizio Affer1, Alejandro Pardo1, Gabriel Pardo1, Justin Taylor1 1Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine The present protocol offers an efficient and flexible method to isolate RNA from nuclear and cytoplasmic fractions using cultured cells, and then validate using qPCR. This effectively serves as a replacement for other RNA preparation kits. Medicine High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model Antoine J. Ribieras1, Yulexi Y. Ortiz1,2, Sophie Shrestha3, C. Theodore Huerta1, Hongwei Shao1,2, Maria E. Boulina4, Roberto I. Vazquez-Padron1,2, Zhao-Jun Liu1,2, Omaida C. Velazquez1,2 1Dewitt Daughtry Family Department of Surgery, University of Miami Miller School of Medicine, 2Vascular Biology Institute, University of Miami Miller School of Medicine, 3University of Miami, 4Analytical Imaging Core Facility, University of Miami The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature. Medicine Electroretinogram Recording for Infants and Children under Anesthesia to Achieve Optimal Dark Adaptation and International Standards Byron L. Lam1, Carlos Mendoza-Santiestaban1, Alex Gonzalez1,2, Cornelis Rowaan1,2, Mu Liu1, Joanne Martin1, Steven Gayer1,3, Osmany Gil Figueredo1, Jean-Marie Parel1,2 1Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 2Ophthalmic Biophysics Center, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 3Department of Anesthesiology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine Adhering to international standards and maintaining retinal dark adaptation are critical to acquire valid full-field electroretinogram responses in the diagnosis and management of inherited retinal diseases. A practical protocol using a portable darkroom is provided to obtain full-field electroretinogram for infants and children under sedation or general anesthesia in the operating room setting. Developmental Biology A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons Amir Bagheri1,2, Seyedeh Fatemeh Razavipour3, Claes Wahlestedt2, Seyed Javad Mowla1, Mohammad Ali Faghihi2,4 1Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, 2Center for Therapeutic Innovation and Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, 3Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, 4Persian BayanGene Research and Training Center The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds. Cancer Research A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery Hongwei Shao1, Mecker Moller1, Dazhi Wang1, Albert Ting1, Marcia Boulina2, Zhao-Jun Liu1 1Department of Surgery, University of Miami School of Medicine, 2Analytical Imaging Core Facility, University of Miami School of Medicine A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics. Medicine A Computerized Functional Skills Assessment and Training Program Targeting Technology Based Everyday Functional Skills Philip D. Harvey1,2, Lize Tibiriçá2,3, Peter Kallestrup2, Sara J. Czaja1,2,4 1University of Miami Miller School of Medicine, 2i-Function, 3Albizu University, 4Weill-Cornell Medical Center This training protocol uses computerized training to teach technology-related everyday functional skills. These skills include financial skills, travel and transit, as well as medication management. Immunology and Infection Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E Duneia McManus1,2,3,4, Horacio J. Novaira1,2,3,4, Anouk A.J. Hamers1,2,3,4, Asha B. Pillai1,2,3,4,5 1Department of Pediatrics, Division of Hematology / Oncology and Bone Marrow Transplantation, University of Miami Miller School of Medicine, 2Batchelor Children's Research Institute, University of Miami Miller School of Medicine, 3Department of Microbiology & Immunology, University of Miami Miller School of Medicine, 4Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, 5Holtz Children's Hospital, University of Miami Miller School of Medicine The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping. Immunology and Infection Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses Petoria Gayle1, Nancy E. Freitag2, Natasa Strbo1, Kurt Schesser1 1Department of Microbiology & Immunology, University of Miami Miller School of Medicine, 2Department of Microbiology & Immunology, University of Illinois College of Medicine We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors. Neuroscience Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue Karolina J. Janczura1,2, Claude-Henry Volmar1,2, Claes Wahlestedt1,2 1Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, 2Center for Therapeutic Innovation, University of Miami Miller School of Medicine The purpose of this article is to provide a comprehensive, systematic guide to the efficient purification of histones H3 and H4 and the quantification of acetylated histone residues. Medicine Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy Eriko Katsuta1, Masanori Oshi1, Omar M. Rashid2,3,4,5, Kazuaki Takabe1,6,7,8,9,10 1Breast Surgery, Department of Surgical Oncology, Roswell Park Comprehensive Cancer Center, 2Holy Cross Hospital Michael and Dianne Bienes Comprehensive Cancer Center, 3Department of Surgery, Massachusetts General Hospital, 4Department of Surgery, University of Miami Miller School of Medicine, 5Department of Surgery, Nova Southeastern University School of Medicine, 6Department of Surgery, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, 7Department of Breast Surgery and Oncology, Tokyo Medical University, 8Department of Surgery, Yokohama City University, 9Department of Surgery, Niigata University Graduate School of Medical and Dental Sciences, 10Department of Surgery, Fukushima Medical University We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression. Neuroscience Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ Jennifer M. Brazill1, Yi Zhu1, Chong Li1, R. Grace Zhai1,2 1Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, 2School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration. Biology Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells Wen Ding1,2, Keyvan Yousefi1,2, Lina A. Shehadeh2,3,4,5 1Department of Molecular and Cellular Pharmacology, University of Miami Leonard M. Miller School of Medicine, 2Interdisciplinary Stem Cell Institute, University of Miami Leonard M. Miller School of Medicine, 3Department of Medicine, Division of Cardiology, University of Miami Leonard M. Miller School of Medicine, 4Vascular Biology Institute, University of Miami Leonard M. Miller School of Medicine, 5Peggy and Harold Katz Family Drug Discovery Center, University of Miami Leonard M. Miller School of Medicine This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics. Behavior Utilizing the Modified T-Maze to Assess Functional Memory Outcomes After Cardiac Arrest Celeste Y. C. Wu1,2, Francesca M. Lerner4, Alexandre Couto e Silva3, Harlee E. Possoit1,2, Tsung-Han Hsieh1,2, Jake T. Neumann5, Alireza Minagar1, Hung Wen Lin1,2,3, Reggie H. C. Lee1,2 1Department of Neurology, Louisiana State University Health Science Center, 2Center for Brain Health, Louisiana State University Health Science Center, 3Department of Cellular Biology and Anatomy, Louisiana State University Health Science Center, 4Department of Neurology, Cerebral Vascular Disease Research Laboratories, University of Miami Miller School of Medicine, 5Department of Biomedical Sciences, West Virginia School of Osteopathic Medicine This protocol describes the use of a modified T-maze to evaluate functional learning/memory in asphyxia cardiac arrest-induced cerebral ischemia. Medicine Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap Yee-Shuan Lee1, Siliang Wu2, Treena Livingston Arinzeh3, Mary Bartlett Bunge1,4,5 1The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, 2Department of Materials Science and Engineering, New Jersey Institute of Technology, 3Department of Biomedical Engineering, New Jersey Institute of Technology, 4Department of Cell Biology, University of Miami Miller School of Medicine, 5Department of Neurological Surgery, University of Miami Miller School of Medicine This article describes a technique to insert a hollow conduit between the spinal cord stumps after complete transection and fill with Schwann cells (SCs) and injectable basement membrane matrix in order to bridge and promote axon regeneration across the gap. Immunology and Infection Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice Brian Czaya*1,2, Saurav Singh*1,2, Christopher Yanucil1,2, Karla Schramm1,2, Christian Faul1,2, Alexander Grabner1 1Katz Family Drug Discovery Center and Division of Nephrology and Hypertension, Department of Medicine, University of Miami Leonard M. Miller School of Medicine, 2Department of Cell Biology and Anatomy, University of Miami Leonard M. Miller School of Medicine Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR. Immunology and Infection Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells Natasa Strbo1, Laura Romero1, Maria Alcaide2, Margaret Fischl2 1Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, 2Division of Infectious Diseases, Miller School of Medicine, University of Miami We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting. Biology Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells Nathalie Baumlin-Schmid1, Matthias Salathe1, Nevis L. Fregien2 1Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, University of Miami, Miller School of Medicine, 2Department of Cell Biology, University of Miami, Miller School of Medicine Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium. Medicine Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy Yan Wang1,3,4, Dale P. Brown1, Brant D. Watson2, Jeffrey L. Goldberg1,3 1Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 2Departments of Neurology and Biomedical Engineering, University of Miami Miller School of Medicine, 3Shiley Eye Center, University of California, 4Department of Ophthalmology and Vision Science, Fudan University The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases. Behavior Development of a Virtual Reality Assessment of Everyday Living Skills Stacy A. Ruse1, Vicki G. Davis1, Alexandra S. Atkins1, K. Ranga R. Krishnan2,3, Kolleen H. Fox4, Philip D. Harvey5, Richard S.E. Keefe1,3 1NeuroCog Trials, Inc., 2Duke-NUS Graduate Medical Center, 3Duke University Medical Center, 4Fox Evaluation and Consulting, PLLC, 5University of Miami Miller School of Medicine A challenge for proving treatment efficacy for cognitive impairments in schizophrenia is finding the optimizing measurement of skills related to everyday functioning. The Virtual Reality Functional Capacity Assessment Tool (VRFCAT) is an interactive gaming based computerized measure aimed at skills associated with everyday functioning, including baseline impairments and treatment related changes.
