University of Oklahoma Health Sciences Center 14 articles published in JoVE Immunology and Infection Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils Sunitha Pulikkot1, Meng Zhao2,3, Zhichao Fan1 1Department of Immunology, School of Medicine, UConn Health, 2Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation, 3Department of Microbiology and Immunology, University of Oklahoma Health Science Center We describe stepwise protocols measuring the mitochondrial respiration of mouse and human neutrophils and HL60 cells using the metabolic extracellular flux analyzer. Medicine Hemodynamic Precision in the Neonatal Intensive Care Unit using Targeted Neonatal Echocardiography Marjorie Makoni*1, Trassanee Chatmethakul*1,2, Regan Giesinger2, Patrick J. McNamara2 1Department of Pediatrics, Division of Neonatal-Perinatal Medicine, University of Oklahoma Health Sciences Center, 2University of Iowa Department of Pediatrics, Division of Neonatology, University of Iowa Presented here is a protocol to perform comprehensive neonatal echocardiography by trained neonatologists in the neonatal intensive care unit. The trained individuals provide longitudinal assessments of heart function, systemic and pulmonary hemodynamics in a consultative role. The manuscript also describes the requirements to become a fully trained neonatal hemodynamics specialist. Medicine Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers Adam Wilson*1, Kathryn Burge*1, Jeffrey Eckert1, Hala Chaaban1 1Department of Pediatrics, Division of Neonatal-Perinatal Medicine, University of Oklahoma Health Sciences Center This protocol describes a method to establish and perform a scratch wound assay on two-dimensional (2D) monolayers derived from three-dimensional (3D) enteroids isolated from non-human primate ileum. Immunology and Infection Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out Enteroid Model Heather Liebe1, Camille Schlegel2, Xue Cai2, Alena Golubkova1, Tyler Leiva1, William L. Berry3, Catherine J. Hunter1 1 The present protocol outlines a method that utilizes lucifer yellow in an apical-out enteroid model to determine intestinal permeability. This method can be used to determine paracellular permeability in enteroids that model inflammatory bowel diseases such as necrotizing enterocolitis. Medicine In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis Kathryn Burge1, Adam Wilson1, Hala Chaaban1 1Department of Pediatrics, Division of Neonatal-Perinatal Medicine, University of Oklahoma Health Sciences Center and Center for Pregnancy and Newborn Health This protocol describes an apical-out necrotizing enterocolitis (NEC)-in-a-dish model utilizing small intestinal enteroids with reversed polarity, allowing access to the apical surface. We provide an immunofluorescent staining protocol to detect NEC-related epithelial disruption and a method to determine the viability of apical-out enteroids subjected to the NEC-in-a-dish protocol. Immunology and Infection Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis Md Huzzatul Mursalin*1,2, Erin Livingston*1, Phillip S. Coburn2,3, Frederick C. Miller4, Roger Astley2,3, Michelle C. Callegan1,2,3 1Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, 2Department of Ophthalmology, Dean McGee Eye Institute, 3Dean McGee Eye Institute, 4Department of Cell Biology and Department of Family and Preventive Medicine, University of Oklahoma Health Sciences Center We describe here a method of intravitreal injection and subsequent bacterial quantitation in mouse model of bacterial endophthalmitis. This protocol can be extended for measuring host immune responses and bacterial and host gene expression. Immunology and Infection Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii Melina B. Cian*1, Nicole P. Giordano*1, Joshua A. Mettlach*1, Keaton E. Minor*1, Zachary D. Dalebroux1 1Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center Gram-negative bacteria produce two spatially segregated membranes. The outer membrane is partitioned from the inner membrane by a periplasm and a peptidoglycan layer. The ability to isolate the dual bilayers of these microbes has been critical for understanding their physiology and pathogenesis. Medicine Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish Jessica Burroughs-Garcia1, Ameera Hasan*1, Gilseung Park*1, Chiara Borga1, J. Kimble Frazer1 1Section of Pediatric Hematology-Oncology, Department of Pediatrics, University of Oklahoma Health Sciences Center Transgenic lck:eGFP zebrafish express GFP highly in T lymphocytes, and have been used to study T cell development and acute lymphoblastic leukemia. This line can be used to study B cells, which express lck at lower levels. This protocol describes purification of malignant and non-malignant B cells from lck:eGFP zebrafish. Medicine Creation of Colonic Anastomosis in Mice Cullen K McCarthy1, Paul K McGaha1, Noah S Rozich1, Nathaniel A Yokell1, Jason S Lees1, William L Berry1 1Department of Surgery, University of Oklahoma Health Sciences Center Anastomotic leak or breakdown after surgery is a major cause of postoperative morbidity and mortality. Our surgery for creating a colonic anastomosis is a reliable and reproducible method for studying the healing mechanisms of anastomoses. Genetics Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System Joseph C. Siefert1,2, Emily A. Clowdus1,2, Duane Goins1, Amnon Koren3, Christopher L. Sansam1,2 1Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, 2Department of Cell Biology, University of Oklahoma Health Sciences Center, 3Department of Molecular Biology and Genetics, Cornell University Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species. Developmental Biology Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea Rabab Sharif1, Shrestha Priyadarsini2, Tyler G. Rowsey2, Jian-Xing Ma3, Dimitrios Karamichos1,2 1Department of Cell Biology, University of Oklahoma Health Sciences Center, 2Department of Ophthalmology/Dean McGee Eye Institute, University of Oklahoma Health Sciences Center, 3Department of Physiology, Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center This protocol describes a novel three-dimensional in vitro model, where corneal stromal cells and differentiated neuronal cells are cultured together to assist in the examination and understanding of the interactions of the two cell types. Genetics An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants Yuhui Chen1, Xianfu Wang2, Shunfei Lu3, Hongcheng Wang2, Shibo Li2, Rujin Chen1 1Laboratory of Plant Genetics and Development, Noble Research Institute, 2Genetics Laboratory, University of Oklahoma Health Science Center, 3Medicine and Health School, Li Shui University This protocol provides experimental steps and information about reagents, equipment, and analysis tools for researchers who are interested in carrying out whole genome array-based comparative genomic hybridization (CGH) analysis of copy number variations in plants. Medicine Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals Phillip G. Durham1, Shumaila N. Hanif2, Lucia Garcia Contreras2, Ellen F. Young3, Miriam S. Braunstein3, Anthony J. Hickey1 1RTI International, 2Oklahoma University Health Science Center, 3University of North Carolina at Chapel Hill During development of drugs for pulmonary delivery, it is necessary to evaluate pharmacokinetics and efficacy in an animal model. We present a method to build a disposable aerosol dispersion system from of-the-shelf components that can be used to administer intrapulmonary dry powder aerosol to rodents. Immunology and Infection Concurrent Quantification of Cellular and Extracellular Components of Biofilms Sharukh S. Khajotia1, Kristin H. Smart1, Mpala Pilula1,3, David M. Thompson2 1Department of Dental Materials, College of Dentistry, University of Oklahoma Health Sciences Center, 2Department of Biostatistics and Epidemiology, College of Public Health, University of Oklahoma Health Sciences Center, 3Department of Biological Sciences, School of Mathematics and Natural Sciences, The Copperbelt University A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.
