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University of Arizona, Tucson

11 ARTICLES PUBLISHED IN JoVE

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Biology

Measuring Caenorhabditis elegans Life Span on Solid Media
George L. Sutphin 1,2, Matt Kaeberlein 1
1Department of Pathology, University of Washington, 2Molecular and Cellular Biology Program, University of Washington

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

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Biology

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer
Huy Nguyen 1, Cristy Loustaunau 1, Alexander Facista 1, Lois Ramsey 1, Nadia Hassounah 1, Hilary Taylor 1, Robert Krouse 2,3, Claire M. Payne 1,4, V. Liana Tsikitis 3, Steve Goldschmid 5, Bhaskar Banerjee 5, Rafael F. Perini 5, Carol Bernstein 1
1Department of Cell Biology and Anatomy, College of Medicine, University of Arizona, Tucson, 2Southern Arizona Veterans Affairs Health Care System, Tucson, AZ, 3Department of Surgery, College of Medicine, University of Arizona, Tucson, 4Biomedical Diagnostics and Research, Tucson, AZ, 5Department of Medicine, College of Medicine, University of Arizona, Tucson

Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.

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Biology

Isolation of Mouse Coronary Endothelial Cells
Shizhen Luo 1, Aaron H. Truong 1, Ayako Makino 1
1Department of Physiology, University of Arizona, Tucson

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

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Immunology and Infection

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration
Emma Teal 1, Nina G. Steele 2, Jayati Chakrabarti 1, Loryn Holokai 3, Yana Zavros 1
1Department of Pharmacology and Systems Physiology, University of Cincinnati, 2Department of Cell & Developmental Biology, University of Michigan, 3Department of Molecular Genetics, Biochemistry & Microbiology, University of Cincinnati

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

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Neuroscience

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels
Hrvoje Brzica *1, Wazir Abdullahi *1, Bianca G. Reilly 1, Patrick T. Ronaldson 1
1Department of Pharmacology, College of Medicine, University of Arizona, Tucson

Here, a method for isolation of rat brain microvessels and for the preparation of membrane samples is described. This protocol has the clear advantage of producing enriched microvessel samples with acceptable protein yield from individual animals. Samples can then be used for robust protein analyses at the brain microvascular endothelium.

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Medicine

A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies
Jayati Chakrabarti *1, Vivien Koh *2,3, Jimmy Bok Yan So 2,4, Wei Peng Yong 2,3, Yana Zavros 1
1Department of Cellular and Molecular Medicine, University of Arizona, Tucson, 2National University Cancer Institute Singapore, National University Health System, Singapore, 3Cancer Science Institute of Singapore, National University of Singapore, 4Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore

The goals of the protocol are to use this approach to 1) understand the role of the immunosuppressive gastric tumor microenvironment and 2) predict the efficacy of patient response, thus increasing the survival rate of patients.

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Neuroscience

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy
Suvithanandhini Loganathan *1, Hannah E. Ball *1, Ernesto Manzo 1,2, Daniela C. Zarnescu 1,3
1Department of Molecular and Cellular Biology, University of Arizona, Tucson, 2Vollum Institute, Oregon Health and Science University, 3Department of Neuroscience, University of Arizona, Tucson

Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

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Behavior

An Inescapable Cat Odor Exposure Protocol for Studying Innate and Contextual Threat Conditioning in Rats
María Rodríguez 1,2, Marco Contreras 3, Marcos Domic-Siede 4, Francisco Ceric 5, Fernando Torrealba 1
1Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, 2Instituto de Ciencias Sociales, Universidad de O'Higgins, 3Department of Psychology, University of Arizona, Tucson, 4Escuela de Psicología, Universidad Católica del Norte, 5Laboratorio de Neurociencia Afectiva, Facultad de Psicología, Universidad del Desarrollo

Cat skin/fur odor exposure provides a reliable method to investigate neural circuits and mechanisms of defensive responses in rodents and may offer insight into mechanisms that mediate fear in humans. Here, we describe a protocol for investigating the role of the interoceptive cortex in threat responses in rats.

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Unfixed Head and Closed Skull Weight Drop Method as a Rodent Model of Mild Traumatic Brain Injury (Mtbi) for Studies of Post-Traumatic Headache (PTH)
Caroline Machado Kopruszinski 1, Trent Anderson 2, Janice Oyarzo 3, Edita Navratilova 1,3, Frank Porreca 1,3
1Department of Pharmacology, College of Medicine, University of Arizona, Tucson, 2Department of Basic Medical Sciences, College of Medicine, University of Arizona, Phoenix, 3Department of Collaborative Research, Mayo Clinic

The protocol describes a rodent model of unfixed head mild traumatic brain injury induced by a weight drop allowing for free linear and rotational impact-related forces. The dynamics of this injury are similar to concussions that occur in humans and are useful to investigate underlying mechanisms involved in post-traumatic headache.

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Developmental Biology

In Vivo Real-Time Study of Drug Effects on Carotid Blood Flow in the Ovine Fetus
Alexander L. Pendleton 1, Sean W. Limesand 1, Ravi Goyal 1
1School of Animal and Comparative Biomedical Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson

The present protocol describes a method to deliver drugs and gene expression-modifying agents perivascularly in an in utero developing fetus. Importantly, the effect of drugs/agents on blood flow can be measured with the progression of pregnancy.

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Biology

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions
Luis Espejo *1, Bradford Hull *1, Leah M. Chang 1, Destiny DeNicola 1, Samuel Freitas 1, Vanessa Silbar 1, Anne Haskins 1, Emily A. Turner 1, George L. Sutphin 1
1Department of Molecular & Cellular Biology, University of Arizona, Tucson

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

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