Queen's University (Kingston, Ontario) View Institution's Website 24 articles published in JoVE Medicine Biventricular Assessment of Cardiac Function and Pressure-Volume Loops by Closed-Chest Catheterization in Mice Francois Potus1, Ashley Y Martin1, Brooke Snetsinger1, Stephen L. Archer1 1 Presented here is a protocol to assess biventricular heart function in mice by generating pressure-volume (PV) loops from the right and left ventricle in the same animal using closed chest catheterization. The focus is on the technical aspect of surgery and data acquisition. Biology Visually Sexing Loggerhead Shrike (Lanius Ludovicianus) Using Plumage Coloration and Pattern Gareth Morgan1, Amy A. Chabot1,2 1African Lion Safari, 2 We present a protocol to characterize the sex of loggerhead shrike visually based on the coloration and pattern of the sixth primary wing feather. Cancer Research Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells Mulu Geletu*1,2, Victoria Hoskin*1, Blerta Starova*1,3, Maximilian Niit1,4, Hanad Adan1, Bruce Elliott1, Patrick Gunning2, Leda Raptis1 1 We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia. Bioengineering A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces Laura A. McKiel1, Kimberly A. Woodhouse1, Lindsay E. Fitzpatrick1 1 This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-кB/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment. Immunology and Infection Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana Melissa Bredow1, Irina Sementchoukova1, Kristen Siegel1, Jacqueline Monaghan1 1 This paper describes two methods for quantifying defense responses in Arabidopsis thaliana following exposure to immune elicitors: the transient oxidative burst, and the inhibition of seedling growth. Biology Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples Alison M. Moore1,2, Lee R. Boudreau3, Shakeel Virk3, David P. LeBrun1,2 1 We describe the use of quantitative immunoblotting to validate immunofluorescence histology coupled with image analysis as a means of quantifying a protein of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Our results demonstrate the utility of immunofluorescence histology for ascertaining the relative quantity of biomarker proteins in routine biopsy samples. Medicine Left Atrial Stenosis Induced Pulmonary Venous Arterialization and Group 2 Pulmonary Hypertension in Rat Ping Yu Xiong*1,2, Shunsuke Baba*3,4, Naritomo Nishioka3,5, Yoshitaka Fujimoto3, Stephen L. Archer1, Susumu Minamisawa1,4 1 Left atrial stenosis (LAS) is a novel surgical technique used for studying group 2 pulmonary hypertension (PH) and mechanisms underlying pulmonary venous arterialization. Here, we present a protocol to constrict the left atrium using a titanium clip to cause pulmonary venous arterialization and moderate PH in a rat. Cancer Research Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor Taha Azad*1, Kazem Nouri*1, Helena J. Janse van Rensburg1, Yawei Hao1, Xiaolong Yang1 1 Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo. Engineering A 3D-printed Chamber for Organic Optoelectronic Device Degradation Testing Emma Mogus1, Benjamin Torres-Kulik1, Christopher Gustin2, Ayse Turak1 1Department of Engineering Physics, McMaster University, 2 Here, we present a protocol for the design, manufacture, and use of a simple, versatile 3D-printed and controlled atmospheric chamber for the optical and electrical characterization of air-sensitive organic optoelectronic devices. 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 Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice Karlaina JL Osmon1, Meera Vyas1, Evan Woodley2, Patrick Thompson2, Jagdeep S Walia1,2,3 1 Behavioral testing is used in pre-clinical trials to assess the phenotypic effects of a disease or treatment on an animal's wellbeing. In order to globally assess motor functioning, we selected tests for general locomotion, muscular strength, and coordination: the open field test, the mesh test, and the rotarod test, respectively. Medicine Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation Alison Michels1, Laura L. Swystun1, Jeffrey Mewburn2, Silvia Albánez1, David Lillicrap1 1 In this paper, we describe a method to assess endothelial von Willebrand factor release and the subsequent platelet capture under fluid shear stress in response to inflammatory stimuli using an in vitro flow chamber system. Neuroscience A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay Jason C. Campbell1, Ian D. Chin-Sang1, William G. Bendena1,2 1 Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state. Biochemistry Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification Melissa Bredow1, Heather E. Tomalty1, Virginia K. Walker1,2,3 1 This paper outlines the identification of ice-binding proteins from freeze-tolerant plants through the assessment of ice-recrystallization inhibition activity and subsequent isolation of native IBPs using ice-affinity purification. 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. Biology An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production Sarah M. Maritan*1,2, Eric Y. Lian*1,2, Lois M. Mulligan1,2 1Department of Pathology and Molecular Medicine, Queen's University Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions. Biology Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction Palak G. Patel1,2, Shamini Selvarajah1,2, Suzanne Boursalie1,2, Nathan E. How1,2, Joshua Ejdelman3, Karl-Philippe Guerard3, John M. Bartlett4, Jacques Lapointe3, Paul C. Park1, John B. A. Okello1,2, David M. Berman1,2 1 This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks. Biology Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis Edmund Ong1,2, Catherine Cahill1,2,3 1Department of Anesthesiology and Perioperative Care, University of California Irvine, 2 Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis. Environment Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites Mackenzie J. Denyes1, Michèle A. Parisien1, Allison Rutter2, Barbara A. Zeeb1 1Department of Chemistry and Chemical Engineering, Royal Military College of Canada, 2 Biochar is a carbon-rich material used as a soil amendment with the ability to sustainably sequester carbon, improve substrate quality and sorb contaminants. This protocol describes the 17 analytical methods used for the characterization of biochar, which is required prior to large scale implementation of these amendments in the environment. Biology A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide Mulu Geletu1, Stephanie Guy1, Kevin Firth2, Leda Raptis1 1 This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide. Chemistry Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity Koli Basu1, Christopher P. Garnham2, Yoshiyuki Nishimiya3, Sakae Tsuda3, Ido Braslavsky4, Peter Davies1 1 Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light. Biology C. elegans Chemotaxis Assay Olivia Margie1, Chris Palmer2, Ian Chin-Sang3 1Life Sciences, Queen's University, 2Department of Chemical Engineering, Queen's University, 3Department of Biology, Queen's University A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest. Biology Mechanical Stimulation of Chondrocyte-agarose Hydrogels James A. Kaupp1, Joanna F. Weber1, Stephen D. Waldman1,2 1Department of Mechanical and Materials Engineering, Queen's University, 2Department of Chemical Engineering, Queen's University The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism. Immunology and Infection Detection of Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation Schammim R. Amith*1, Preethi Jayanth*1, Trisha Finlay*1, Susan Franchuk*1, Alanna Gilmour*1, Samar Abdulkhalek1, Myron R. Szewczuk*1 1Department of Microbiology and Immunology, Queen's University - Kingston, Ontario The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.