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University of Western Ontario

22 ARTICLES PUBLISHED IN JoVE

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Medicine

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging
Choi-Fong Cho 1, Amber Ablack 2, Hon-Sing Leong 2, Andries Zijlstra 3, John Lewis 2,4
1Department of Medical Biophysics, University of Western Ontario, 2London Regional Cancer Program, London Health Science Centre, 3Department of Pathology, Vanderbilt University , 4Translational Prostate Cancer Research Group, London Health Science Centre

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

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Biology

Evaluation of Mammary Gland Development and Function in Mouse Models
Isabelle Plante 1, Michael K.G. Stewart 1, Dale W. Laird 1
1Department of Anatomy and Cell Biology, University of Western Ontario

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

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Neuroscience

Habituation and Prepulse Inhibition of Acoustic Startle in Rodents
Bridget Valsamis 1, Susanne Schmid 1
1Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario

Habituation and prepulse inhibition of startle are operational measures of sensory gating. Sensory gating is disrupted in schizophrenia, and some other mental disorders and neurodegenerative diseases. We here describe a standard protocol to assess short-term and long-term habituation as well as prepulse inhibition of acoustic startle responses in rats and mice.

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Biology

Growth Assays to Assess Polyglutamine Toxicity in Yeast
Martin L. Duennwald 1
1Boston Biomedical Research Institute

This manuscript describes three complementary protocols for assessing the toxicity of polyglutamine (polyQ)-expansion proteins in the yeast Saccharomyces cerevisiae. These protocols can easily be modified to monitor the toxicity of other misfolded proteins in yeast.

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Biology

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System
Gang Tian 1, Qing Lu 2, Li Zhang 2, Susanne E. Kohalmi 1, Yuhai Cui 2
1Department of Biology, University of Western Ontario, 2Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

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JoVE Journal

Analysis of Cell Cycle Position in Mammalian Cells
Matthew J. Cecchini 1, Mehdi Amiri 1, Frederick A. Dick 2
1Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, 2London Regional Cancer Program, Children's Health Research Institute, and Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

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Neuroscience

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI
Trecia A. Brown 1,6, Joseph S. Gati 5, Sarah M. Hughes 5, Pam L. Nixon 1,6, Ravi S. Menon 3,4,5, Stephen G. Lomber 1,2,4,5,6,7
1Department of Physiology and Pharmacology, University of Western Ontario, 2Department of Psychology, University of Western Ontario, 3Department of Medical Biophysics, University of Western Ontario, 4Brain and Mind Institute, University of Western Ontario, 5Centre for Functional and Metabolic Mapping, Robarts Research Institute, University of Western Ontario, 6Cerebral Systems Laboratory, University of Western Ontario, 7National Centre for Audiology, University of Western Ontario

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

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Behavior

A Method for Investigating Age-related Differences in the Functional Connectivity of Cognitive Control Networks Associated with Dimensional Change Card Sort Performance
Bianca DeBenedictis 1, J. Bruce Morton 1
1Cognitive Development and Neuroimaging Laboratory, Department of Psychology, Brain and Mind Institute, University of Western Ontario

This video presents a method of examining age-related changes in functional connectivity of cognitive control networks engaged by targeted tasks/processes. The technique is based on multi-variate analysis of fMRI data.

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Medicine

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model
Donna Cvetković *1, Cameron Glenn-Franklin Goertzen *1, Moshmi Bhattacharya 1,2,3
1Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, 2Department of Oncology, Schulich School of Medicine and Dentistry, University of Western Ontario, 3Lawson Health Research Institute

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

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Developmental Biology

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus
Steven J. Deimling 1, Rami R. Halabi 2,3, Stephanie A. Grover 4, Jean H. Wang 2,5, Thomas A. Drysdale 2,4,5
1Developmental and Stem Cell Biology, Hospital for Sick Children, 2Children's Health Research Institute, University of Western Ontario, 3Department of Physiology and Pharmacology, University of Western Ontario, 4Neurosciences and Mental Health, Hospital for Sick Children, 5Department of Paediatrics, University of Western Ontario

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

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Neuroscience

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster
Robert W. Fernandez 1, Marat Nurilov 2, Omar Feliciano 2, Ian S. McDonald 3, Anne F. Simon 3
1Department of Molecular Biophysics and Biochemistry, Yale University, 2Department of Biology, York College/CUNY, 3Department of Biology, Western Ontario University

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

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Bioengineering

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration
Sébastien Meghezi 1, Dawit G. Seifu 1,2, Nina Bono 1,3, Larry Unsworth 2,4,5, Kibret Mequanint 6, Diego Mantovani 1,2
1Laboratory for Biomaterials and Bioengineering, Department Min-Met-Materials Eng & CHU de Québec Research Center, Canada Research Chair I for the Innovation in Surgery, Laval University, 2NSERC CREATE Program for Regenerative Medicine (NCPRM), Laval University, 3Department Electronics, Information and Bioengineering, Politecnico di Milano, 4Department of Chemical and Materials Engineering, University of Alberta, 5National Institute for Nanotechnology, National Research Council (Canada), 6Department of Chemical and Biochemical Engineering, University of Western Ontario

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

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Neuroscience

Conditions Affecting Social Space in Drosophila melanogaster
Alison R. McNeil 1, Sam N. Jolley 1, Adesanya A. Akinleye 2, Marat Nurilov 2, Zulekha Rouzyi 2, Austin J. Milunovich 3, Moria C. Chambers 3, Anne F. Simon 1
1Department of Biology, University of Western Ontario, 2Department of Biology, York College/CUNY, 3Department of Entomology, Cornell University

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

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Medicine

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration
Jessica N. Blom 1, Xiangru Lu 1, Paul Arnold 1, Qingping Feng 1
1Department of Physiology and Pharmacology, Western University

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

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Developmental Biology

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands
Elham Dianati 1,2, Isabelle Plante 1
1INRS, Institut Armand-Frappier, 2Biomed, UQAM

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

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JoVE Journal

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling
Naeem Nathoo 1,2, Mark A. Bernards 2, Jacqueline MacDonald 3, Ze-Chun Yuan 1,3
1London Research and Development Centre, Agriculture & Agri-Food Canada, 2Department of Biology, University of Western Ontario, 3Department of Microbiology and Immunology, University of Western Ontario

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

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Behavior

Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat
Carmen Wong 1,2, Stephen G. Lomber 1,2,3,4,5
1Cerebral Systems Laboratory, University of Western Ontario, 2Graduate Program in Neuroscience, University of Western Ontario, 3Brain and Mind Institute, University of Western Ontario, 4Department of Physiology and Pharmacology, University of Western Ontario, 5Department of Psychology, University of Western Ontario

Complex locomotion in naturalistic environments requiring careful coordination of the limbs involves regions of the parietal cortex. The following protocol describes the use of reversible cooling-induced deactivation to demonstrate the role of parietal area 5 in memory-guided obstacle avoidance in the walking cat.

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Immunology and Infection

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins
Yoo Jung Choi *1, Jinhui Zhu *1, Steve Chung *1, Naveed Siddiqui *1, Qingping Feng 1, Peter B. Stathopulos 1
1Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

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Education

Esophageal Heat Transfer for Patient Temperature Control and Targeted Temperature Management
Melissa I. Naiman 1,2, Maria Gray 2, Joseph Haymore 3, Ahmed F. Hegazy 4, Andrej Markota 5, Neeraj Badjatia 6, Erik B. Kulstad 2,7
1Center for Advanced Design, Research, and Exploration, University of Illinois at Chicago, 2Attune Medical, 3University of Maryland School of Nursing, 4University of Western Ontario, 5University Medical Centre Maribor, 6University of Maryland, 7Department of Emergency Medicine, University of Texas, Southwestern Medical Center

This study presents a novel method to provide efficient patient temperature control for cooling or warming patients. A single use, triple lumen device is placed into the esophagus, analogous to a standard orogastric tube, and connects to existing heat exchange units to perform automatic patient temperature management.

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Immunology and Infection

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy
Kyle Taruc 1, Charles Yin 1, Daniel G. Wootton 2,3, Bryan Heit 1
1Department of Microbiology and Immunology and the Center for Human Immunology, University of Western Ontario, 2Institute of Infection and Global Health, University of Liverpool, 3Department of Respiratory Research, Aintree University Hospital NHS Foundation Trust

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

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Biology

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast
Yuwei Jiang 1, Sonja Di Gregorio 2, Maram B. Albakri 1, Martin L. Duennwald 1,2, Patrick Lajoie 1
1Department of Anatomy and Cell Biology, University of Western Ontario, 2Department of Pathology and Laboratory Medicine, University of Western Ontario

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

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Neuroscience

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology
Julia K. Sunstrum 1, Wataru Inoue 1,2,3
1Neuroscience Program, Schulich School of Medicine and Dentistry, University of Western Ontario, 2Robarts Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, 3Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

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