Midwestern University 12 articles published in JoVE Immunology and Infection Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research Deborah Molehin1, Jack Guinan1, Brina Lopez1 1Department of Farm Animal Medicine, College of Veterinary Medicine, Midwestern University Enteroids are emerging as a novel model for studying tissue physiology and pathophysiology, drug development, and regenerative medicine. Here, we describe a bovine primary cell 2D enteroid-derived culture system that permits co-culture with relevant tissue cell types. This model offers a translational advantage for gastrointestinal research modeling. Biology Measurement of Endothelium-Dependent Vasorelaxation in the Mouse Thoracic Aorta Using Tensometric Small Volume Chamber Myography Brikena Gusek1, Robert Folk1, Tala Curry2, Mitra Esfandiarei1 1Department of Biomedical Sciences, College of Graduate Studies, Midwestern University, 2Department of Child Health, College of Medicine-Phoenix, University of Arizona, Phoenix The present protocol describes the concepts and technical application of the tensometric myograph technique using a multi-chamber myograph system in the experimental ex vivo assessment of mouse aortic endothelial function. Immunology and Infection An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract Ross Monasky1, Sonia Villa2, Shankar Thangamani3 1College of Veterinary Medicine, Midwestern University, 2Masters in Biomedical Science Program, College of Graduate Studies, Midwestern University, 3Department of Pathology and Population Medicine, College of Veterinary Medicine, Midwestern University The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut. Immunology and Infection Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis Jesse Q. Nguyen1, Xhavit Zogaj1, Aanuoluwa A. Adelani1, Ping Chu2, Jieh-Juen Yu1, Bernard P. Arulanandam1, Karl E. Klose1 1South Texas Center for Emerging Infectious Diseases, University of Texas San Antonio, 2Midwestern University This protocol describes intratracheal inoculations of Fischer 344 rats with Francisella tularensis. This procedure mimics pulmonary exposure of humans to this potential biothreat agent and can be used to test vaccine and therapeutic efficacy against pulmonary tularemia. Behavior Creating and Applying a Reference to Facilitate the Discussion and Classification of Proteins in a Diverse Group D. Ellen K. Tarr1 1Department of Microbiology and Immunology, Arizona College of Osteopathic Medicine, Midwestern University The goal of this protocol is to develop a reference for divergent proteins in a group that lacks coherent criteria for nomenclature and classification. This reference will facilitate analyses and discussion of the group as a whole and can be used in addition to established names. Medicine Establishment of a Clinic-based Biorepository Sarah E. Belden1, Chandana K. Uppalapati2, Agnes S. Pascual3, McKale R. Montgomery3, Kathryn J. Leyva2, Elizabeth E. Hull3, Richard L. Averitte1 1Affiliated Dermatology & Affiliated Laboratories, Midwestern University Osteopathic Postdoctoral Training Institute, Midwestern University, 2Department of Microbiology & Immunology, Arizona College of Osteopathic Medicine, Midwestern University, 3Biomedical Sciences Program, College of Health Sciences, Midwestern University Cutaneous tumors are often discarded following Mohs micrographic surgery. A protocol is described here that enables clinical support staff to effectively process and store cutaneous tumor (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) samples for downstream laboratory applications without interfering with clinical operations. Biology Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts Yuriana Aguilar-Sanchez*1, Diego Fainstein*2,3, Rafael Mejia-Alvarez4, Ariel L. Escobar5 1School of Natural Sciences, University of California, Merced, 2Centro de Investigaciones Cardiovasculares, Universidad de la Plata and Conicet, 3Facultad de Ingenieria, Universidad Nacional de Entre Rios, 4Department of Physiology, Midwestern University, 5School of Engineering, University of California, Merced In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations. Immunology and Infection Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi Balbina J. Plotkin1, Ira M. Sigar1, Vaibhav Tiwari1, Scott Halkyard1 1Department of Microbiology and Immunology, Midwestern University A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses. Biology Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging Gabriela Ziomek1, Cornelis van Breemen1, Mitra Esfandiarei1,2 1Department of Anaesthesiology, Pharmacology, and Therapeutics, University of British Columbia, 2Department of Biomedical Sciences, Midwestern University Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen. Developmental Biology Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing Jose L. Rapanan1, Agnes S. Pascual1, Chandana K. Uppalapati2, Kimbal E. Cooper1, Kathryn J. Leyva2, Elizabeth E. Hull1 1Biomedical Sciences Program, Midwestern University, 2Department of Microbiology & Immunology, Midwestern University Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays. Behavior Training Rats to Voluntarily Dive Underwater: Investigations of the Mammalian Diving Response Paul F. McCulloch1 1Department of Physiology, Midwestern University Detailed instructions are provided on how to train rats to voluntarily dive underwater through a 5 m long Plexiglas maze. Because the brains of rats have been very well characterized, voluntarily diving rats may help elucidate the central pathways of the mammalian diving response. Biology Dissection and Mounting of Drosophila Pupal Eye Discs Joy S. Tea1, Albert Cespedes1, Daniel Dawson2, Utpal Banerjee1,3, Gerald B. Call2 1Department of Molecular, Cell and Developmental Biology (MCDB), University of California at Los Angeles (UCLA), 2Arizona College of Osteopathic Medicine (AZCOM), Midwestern University, 3MCDB, Broad Stem Cell Research Center, UCLA The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.
