Western University View Institution's Website 29 articles published in JoVE Neuroscience A Standardized Pipeline for Examining Human Cerebellar Grey Matter Morphometry using Structural Magnetic Resonance Imaging Rebecca Kerestes1, Shuo Han2, Srinivas Balachander3, Carlos Hernandez-Castillo4, Jerry L. Prince5,6, Jörn Diedrichsen7, Ian H. Harding1,8 1Department of Neuroscience, Central Clinical School, Monash University, 2Department of Biomedical Engineering, The Johns Hopkins University, 3Department of Psychiatry, National Institute of Mental Health & Neuro Sciences (NIMHANS), 4Faculty of Computer Science, Dalhousie University, 5Department of Electrical and Computer Engineering, The Johns Hopkins University, 6Department of Computer Science, The Johns Hopkins University, 7Brain and Mind Institute, Department for Statistical and Actuarial Sciences, Department for Computer Science, Western University, 8Monash Biomedical Imaging, Monash University A standardized pipeline is presented for examining cerebellum grey matter morphometry. The pipeline combines high-resolution, state-of-the-art approaches for optimized and automated cerebellum parcellation and voxel-based registration of the cerebellum for volumetric quantification. Cancer Research Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples Vasudeva Bhat1,2, Cory Lefebvre1,2, David Goodale1, Mauricio Rodriguez-Torres1,2, Alison L. Allan1,2,3,4 1London Regional Cancer Program, 2Department of Anatomy & Cell Biology, Western University, 3Department of Oncology, Western University, 4Lawson Health Research Institute This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function. Neuroscience An Emerging Target Paradigm to Evoke Fast Visuomotor Responses on Human Upper Limb Muscles Rebecca A. Kozak1,4, Aaron L. Cecala2,4, Brian D. Corneil1,2,3,4 1Graduate Program in Neuroscience, Western University, 2Department of Physiology and Pharmacology, Western University, 3Department of Psychology, Western University, 4Robarts Research Institute Presented here is a behavioral paradigm that elicits robust fast visuomotor responses on human upper limb muscles during visually guided reaches. Bioengineering Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate Soojin Kim1, Francisco Martinez-Santiesteban2, Timothy J. Scholl1,2,3 1Department of Medical Biophysics, Western University, 2Imaging Research Laboratory, Robarts Research Institute, Western University, 3Ontario Institute for Cancer Research We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates. Immunology and Infection Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses Joshua Choi1, Courtney E. Meilleur1, S.M. Mansour Haeryfar1,2,3,4 1Department of Microbiology and Immunology, Western University, 2Department of Medicine, Division of Clinical Immunology and Allergy, Western University, 3Department of Surgery, Division of General Surgery, Western University, 4Lawson Health Research Institute We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing. Immunology and Infection Quantification of Efferocytosis by Single-cell Fluorescence Microscopy Kyle Taruc1, Charles Yin1, Daniel G. Wootton2,3, Bryan Heit1 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. Genetics Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease Allison A. Dilliott1,2, Sali M.K. Farhan3, Mahdi Ghani4, Christine Sato4, Eric Liang5, Ming Zhang4, Adam D. McIntyre1, Henian Cao1, Lemuel Racacho6,7, John F. Robinson1, Michael J. Strong1,8, Mario Masellis9,10, Dennis E. Bulman6,7, Ekaterina Rogaeva4, Anthony Lang10,11, Carmela Tartaglia4,10, Elizabeth Finger12,13, Lorne Zinman9, John Turnbull14, Morris Freedman10,15, Rick Swartz9, Sandra E. Black9,16, Robert A. Hegele1,2 1Robarts Research Institute, Schulich School of Medicine and Dentistry, Western University, 2Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, 3Analytic and Translational Genetics Unit, Center for Genomic Medicine, Harvard Medical School, Massachusetts General Hospital, Stanley Centre for Psychiatric Research, Broad Institute of MIT and Harvard, 4Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, 5 Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease. Behavior Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat Carmen Wong1,2, Stephen G. Lomber1,2,3,4,5 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. Medicine Esophageal Heat Transfer for Patient Temperature Control and Targeted Temperature Management Melissa I. Naiman1,2, Maria Gray2, Joseph Haymore3, Ahmed F. Hegazy4, Andrej Markota5, Neeraj Badjatia6, Erik B. Kulstad2,7 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. 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 Feng1, Peter B. Stathopulos1 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. Genetics Expedited Radiation Biodosimetry by Automated Dicentric Chromosome Identification (ADCI) and Dose Estimation Ben Shirley*1, Yanxin Li*1, Joan H.M. Knoll1,2, Peter K. Rogan1,3 1CytoGnomix Inc., 2Department of Pathology and Laboratory Medicine, Western University, 3Department of Biochemistry, Western University The cytogenetic dicentric chromosome (DC) assay quantifies exposure to ionizing radiation. The Automated Dicentric Chromosome Identifier and Dose Estimator software accurately and rapidly estimates biological dose from DCs in metaphase cells. It distinguishes monocentric chromosomes and other objects from DCs, and estimates biological radiation dose from the frequency of DCs. Biology A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling Naeem Nathoo1,2, Mark A. Bernards2, Jacqueline MacDonald3, Ze-Chun Yuan1,3 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. Neuroscience Drosophila Courtship Conditioning As a Measure of Learning and Memory Tom S. Koemans1,2,3, Cornelia Oppitz4, Rogier A. T. Donders5, Hans van Bokhoven1,3, Annette Schenck1,3, Krystyna Keleman6, Jamie M. Kramer7,8 1Department of Human Genetics, Radboud University Medical Center, 2Radboud Institute of Molecular Life Sciences, Radboud University, 3Donders Institute for Brain, Cognition, and Behaviour, Centre for Neuroscience, Radboud University, 4Research Institute of Molecular Pathology, Vienna, Austria, 5Department for Health Evidence, Radboud University Medical Center, 6Janelia Research Campus, Howard Hughes Medical Institute, 7Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, 8Department of Biology, Faculty of Science, Western University This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory. Bioengineering Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms Anna Kornmuller1, Cody F.C. Brown2, Claire Yu3, Lauren E. Flynn2,4 1Biomedical Engineering Graduate Program, The University of Western Ontario, 2Department of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, The University of Western Ontario, 3 The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds. Neuroscience Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry Sarah Caughlin1, Dae Hee Park1, Ken K-C Yeung2, David F. Cechetto1, Shawn N. Whitehead1 1Department of Anatomy and Cell Biology, The University of Western Ontario, 2Department of Chemistry and Biochemistry, The University of Western Ontario A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented. Immunology and Infection Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies Rajiv W. Jain1, Amy K. Dang1, Steven M. Kerfoot1 1Department of Microbiology and Immunology, Western University We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells. Medicine Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior Matthew M. Piaseczny*1,2, Graciella M. Pio*1,2, Jenny E. Chu1,2, Ying Xia1, Kim Nguyen1,3, David Goodale1, Alison Allan1,2,4,5 1London Regional Cancer Program, London Health Sciences Centre, 2Department of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, Western University, 3Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, 4Department of Oncology, Schulich School of Medicine & Dentistry, Western University, 5Lawson Health Research Institute This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells. Medicine Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration Jessica N. Blom1, Xiangru Lu1, Paul Arnold1, Qingping Feng1 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. Medicine Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer's Disease with Stroke Jennifer L. Au1, Nina Weishaupt1, Hayley J. Nell1, Shawn N. Whitehead1, David F. Cechetto1 1Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, Western University To investigate the co-morbid Alzheimer's disease (AD) and stroke condition in a novel model, three behavior tasks are described that assess both motor control and cognitive behaviors. These tasks include the beam-walk task, cylinder task and Morris water maze. Biology Imaging the Intracellular Trafficking of APP with Photoactivatable GFP Joshua H. K. Tam1, Stephen H. Pasternak1,2 1Department of Physiology and Pharmacology, Robarts Research Institute, Western University, 2Department of Clinical Neurological Sciences, Western University While the transport of cell surface proteins is relatively easily studied, visualizing the trafficking of intracellular proteins is much more difficult. Here, we use constructs incorporating photoactivatable GFP and demonstrate a method to accurately follow the amyloid precursor protein from the Golgi apparatus to down-stream compartments and follow its clearance. Medicine Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo Mateusz Rytelewski1, Adrian Buensuceso2, Hon S. Leong3, Bonnie J. Deroo2, Ann F. Chambers4, James Koropatnick1 1Microbiology and Immunology, Western University, 2Biochemistry, Western University, 3Surgery, Western University, 4Oncology, Western University Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics. Medicine Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications Lori E. Lowes1,2, Benjamin D. Hedley3, Michael Keeney3,4, Alison L. Allan1,2,4,5 1London Regional Cancer Program, London Health Sciences Centre, 2Department of Anatomy & Cell Biology, Schulich School of Medicine and Dentistry, Western University, 3Special Hematology/Flow Cytometry, London Health Sciences Centre, 4Lawson Health Research Institute, 5Department of Oncology, Western University Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology. Medicine Molecular Imaging to Target Transplanted Muscle Progenitor Cells Kelly Gutpell1,2, Rebecca McGirr1, Lisa Hoffman1,2,3 1Imaging Program, Lawson Health Research Institute, 2Department of Anatomy and Cell Biology, Western University, 3Department of Medical Biophysics, Western University A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging. Biology Single Oocyte Bisulfite Mutagenesis Michelle M. Denomme1,2,3, Liyue Zhang3, Mellissa R.W. Mann1,2,3 1Department of Obstretrics & Gynaecology, Schulich School of Medicine and Dentistry, University of Western Ontario, 2Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, 3Children's Health Research Institute Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos. Biology Analysis of Cell Cycle Position in Mammalian Cells Matthew J. Cecchini1, Mehdi Amiri1, Frederick A. Dick2 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. Biology Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System Gang Tian1, Qing Lu2, Li Zhang2, Susanne E. Kohalmi1, Yuhai Cui2 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. Neuroscience Habituation and Prepulse Inhibition of Acoustic Startle in Rodents Bridget Valsamis1, Susanne Schmid1 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. Biology Evaluation of Mammary Gland Development and Function in Mouse Models Isabelle Plante1, Michael K.G. Stewart1, Dale W. Laird1 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. Medicine Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging Choi-Fong Cho1, Amber Ablack2, Hon-Sing Leong2, Andries Zijlstra3, John Lewis2,4 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.