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Midwestern University

15 ARTICLES PUBLISHED IN JoVE

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Behavior

Training Rats to Voluntarily Dive Underwater: Investigations of the Mammalian Diving Response
Paul F. McCulloch 1
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.

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Biology

Dissection and Mounting of Drosophila Pupal Eye Discs
Joy S. Tea 1, Albert Cespedes 1, Daniel Dawson 2, Utpal Banerjee 1,3, Gerald B. Call 2
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.

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Developmental Biology

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing
Jose L. Rapanan 1, Agnes S. Pascual 1, Chandana K. Uppalapati 2, Kimbal E. Cooper 1, Kathryn J. Leyva 2, Elizabeth E. Hull 1
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.

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Biology

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging
Gabriela Ziomek 1, Cornelis van Breemen 1, Mitra Esfandiarei 1,2
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.

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Immunology and Infection

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi
Balbina J. Plotkin 1, Ira M. Sigar 1, Vaibhav Tiwari 1, Scott Halkyard 1
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.

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Biology

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts
Yuriana Aguilar-Sanchez *1, Diego Fainstein *2,3, Rafael Mejia-Alvarez 4, Ariel L. Escobar 5
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.

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Medicine

Establishment of a Clinic-based Biorepository
Sarah E. Belden 1, Chandana K. Uppalapati 2, Agnes S. Pascual 3, McKale R. Montgomery 3, Kathryn J. Leyva 2, Elizabeth E. Hull 3, Richard L. Averitte 1
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.

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Behavior

Creating and Applying a Reference to Facilitate the Discussion and Classification of Proteins in a Diverse Group
D. Ellen K. Tarr 1
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.

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Immunology and Infection

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis
Jesse Q. Nguyen 1, Xhavit Zogaj 1, Aanuoluwa A. Adelani 1, Ping Chu 2, Jieh-Juen Yu 1, Bernard P. Arulanandam 1, Karl E. Klose 1
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.

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Neuroscience

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection
Michele Fornaro 1, Harsh Sharthiya 1, Vaibhav Tiwari 2
1Department of Anatomy, Chicago College of Osteopathic Medicine (CCOM), Midwestern University, 2Department of Microbiology and Immunology, Chicago College of Osteopathic Medicine (CCOM), Midwestern University

In this report, the advantages of organotypic cultures and dissociated primary cultures of mouse-derived dorsal root ganglia are highlighted to investigate a wide range of mechanisms associated with neuron-glial interaction, neuroplasticity, neuroinflammation, and response to viral infection.

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Biology

Anaerobic Growth and Maintenance of Mammalian Cell Lines
Balbina J. Plotkin 1, Ira M. Sigar 1, Julie A. Swartzendruber 1, Amber Kaminski 1
1Department of Microbiology and Immunology, Midwestern University

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

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Immunology and Infection

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract
Ross Monasky 1, Sonia Villa 2, Shankar Thangamani 3
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.

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Biology

Measurement of Endothelium-Dependent Vasorelaxation in the Mouse Thoracic Aorta Using Tensometric Small Volume Chamber Myography
Brikena Gusek 1, Robert Folk 1, Tala Curry 2, Mitra Esfandiarei 1
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.

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Immunology and Infection

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research
Deborah Molehin 1, Jack Guinan 1, Brina Lopez 1
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.

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JoVE Journal

Modifying Levels of Maternal Dietary Folic Acid or Choline to Study the Impact of Deficiencies on Offspring Health Outcomes
Mitra Esfandiarei 1,2,3, Teodoro Bottiglieri 4, Nafisa M. Jadavji 5,6,7
1Department of Biomedical Sciences, Midwestern University, 2Department of Basic Medical Sciences, College of Medicine Phoenix, University of Arizona, 3Department of Anesthesiology, Pharmacology, & Therapeutics, Faculty of Medicine, University of British Columbia, 4Center of Metabolomics, Institute of Metabolic Disease, Baylor Scott & White Research Institute, 5Department of Physiology, Southern Illinois University, 6Department of Child Health, College of Medicine Phoenix, University of Arizona, 7Department of Neuroscience, Carleton University

This protocol describes the method for reducing dietary intake of folic acid or choline in female mice prior to pregnancy with the objective of investigating the impact of maternal diet on offspring health outcomes.

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