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18 ARTICLES PUBLISHED IN JoVE

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Medicine

Molecular Imaging to Target Transplanted Muscle Progenitor Cells
Kelly Gutpell 1,2, Rebecca McGirr 1, Lisa Hoffman 1,2,3
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.

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Medicine

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications
Lori E. Lowes 1,2, Benjamin D. Hedley 3, Michael Keeney 3,4, Alison L. Allan 1,2,4,5
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.

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Medicine

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo
Mateusz Rytelewski 1, Adrian Buensuceso 2, Hon S. Leong 3, Bonnie J. Deroo 2, Ann F. Chambers 4, James Koropatnick 1
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.

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Medicine

Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer's Disease with Stroke
Jennifer L. Au 1, Nina Weishaupt 1, Hayley J. Nell 1, Shawn N. Whitehead 1, David F. Cechetto 1
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.

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Biology

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP
Joshua H. K. Tam 1, Stephen H. Pasternak 1,2
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.

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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. Chu 1,2, Ying Xia 1, Kim Nguyen 1,3, David Goodale 1, Alison Allan 1,2,4,5
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.

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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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Immunology and Infection

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies
Rajiv W. Jain 1, Amy K. Dang 1, Steven M. Kerfoot 1
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.

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Neuroscience

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry
Sarah Caughlin 1, Dae Hee Park 1, Ken K-C Yeung 2, David F. Cechetto 1, Shawn N. Whitehead 1
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.

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Neuroscience

Drosophila Courtship Conditioning As a Measure of Learning and Memory
Tom S. Koemans 1,2,3, Cornelia Oppitz 4, Rogier A. T. Donders 5, Hans van Bokhoven 1,3, Annette Schenck 1,3, Krystyna Keleman 6, Jamie M. Kramer 7,8
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.

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Genetics

Expedited Radiation Biodosimetry by Automated Dicentric Chromosome Identification (ADCI) and Dose Estimation
Ben Shirley *1, Yanxin Li *1, Joan H.M. Knoll 1,2, Peter K. Rogan 1,3
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.

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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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JoVE Journal

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease
Allison A. Dilliott 1,2, Sali M.K. Farhan 3, Mahdi Ghani 4, Christine Sato 4, Eric Liang 5, Ming Zhang 4, Adam D. McIntyre 1, Henian Cao 1, Lemuel Racacho 6,7, John F. Robinson 1, Michael J. Strong 1,8, Mario Masellis 9,10, Dennis E. Bulman 6,7, Ekaterina Rogaeva 4, Anthony Lang 10,11, Carmela Tartaglia 4,10, Elizabeth Finger 12,13, Lorne Zinman 9, John Turnbull 14, Morris Freedman 10,15, Rick Swartz 9, Sandra E. Black 9,16, Robert A. Hegele 1,2
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, 5School of Medicine, Faculty of Health Sciences, Queen's University, 6Faculty of Medicine, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 7CHEO Research Institute, Faculty of Medicine, University of Ottawa, 8Department of Clinical Neurological Sciences, Western University, 9Division of Neurology, Department of Medicine, Sunnybrook Health Sciences Centre, University of Toronto, 10Division of Neurology, Department of Medicine, University of Toronto, 11Morton and Gloria Shulman Movement Disorders Centre, Toronto Western Hospital, 12Department of Clinical Neurological Sciences, Schulich School of Medicine and Dentistry, Western University, 13Parkwood Institute, St. Joseph's Health Care, 14Department of Medicine, Division of Neurology, McMaster University, 15Division of Neurology, Department of Medicine, Baycrest Health Sciences, 16Canadian Partnership for Stroke Recovery Sunnybrook Site, Sunnybrook Health Science Centre, University of Toronto

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.

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Bioengineering

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate
Soojin Kim 1, Francisco Martinez-Santiesteban 2, Timothy J. Scholl 1,2,3
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.

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Immunology and Infection

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses
Joshua Choi 1, Courtney E. Meilleur 1, S.M. Mansour Haeryfar 1,2,3,4
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.

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Neuroscience

An Emerging Target Paradigm to Evoke Fast Visuomotor Responses on Human Upper Limb Muscles
Rebecca A. Kozak 1,4, Aaron L. Cecala 2,4, Brian D. Corneil 1,2,3,4
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.

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Cancer Research

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples
Vasudeva Bhat 1,2, Cory Lefebvre 1,2, David Goodale 1, Mauricio Rodriguez-Torres 1,2, Alison L. Allan 1,2,3,4
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.

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Neuroscience

A Standardized Pipeline for Examining Human Cerebellar Grey Matter Morphometry using Structural Magnetic Resonance Imaging
Rebecca Kerestes 1, Shuo Han 2, Srinivas Balachander 3, Carlos Hernandez-Castillo 4, Jerry L. Prince 5,6, Jörn Diedrichsen 7, Ian H. Harding 1,8
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.

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