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25 ARTICLES PUBLISHED IN JoVE

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Bioengineering

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells
M. Dean Chamberlain 1, Mark J. Butler 1, Ema C. Ciucurel 1, Lindsay E. Fitzpatrick 2, Omar F. Khan 1, Brendan M. Leung 2, Chuen Lo 1, Ritesh Patel 2, Alexandra Velchinskaya 2, Derek N. Voice 2, Michael V. Sefton 1
1Institute of Biomaterials and Biomedical Engineering / Department of Chemical Engineering and Applied Chemistry, University of Toronto, 2Institute of Biomaterials and Biomedical Engineering, University of Toronto

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

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Chemistry

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity
Koli Basu 1, Christopher P. Garnham 2, Yoshiyuki Nishimiya 3, Sakae Tsuda 3, Ido Braslavsky 4, Peter Davies 1
1Department of Biomedical and Molecular Sciences, Queen's University, 2National Institute of Neurological Disorders and Stroke, Porter Neuroscience Research Center, 3Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Science and Technology, 4The Robert H. Smith Faculty of Agriculture, Food and Environment, Institute of Biochemistry, Food Science, and Nutrition, The Hebrew University of Jerusalem

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.

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Biology

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide
Mulu Geletu 1, Stephanie Guy 1, Kevin Firth 2, Leda Raptis 1
1Department of Microbiology and Immunology and Department of Pathology, Queen's University, 2Ask Science Products Inc.

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.

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Environment

Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites
Mackenzie J. Denyes 1, Michèle A. Parisien 1, Allison Rutter 2, Barbara A. Zeeb 1
1Department of Chemistry and Chemical Engineering, Royal Military College of Canada, 2Analytical Services Unit, School of Environmental Studies, Queen's University

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.

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Biology

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis
Edmund Ong 1,2, Catherine Cahill 1,2,3
1Department of Anesthesiology and Perioperative Care, University of California Irvine, 2Department of Biomedical and Molecular Sciences, Queen's University, 3Department of Pharmacology, University of California Irvine

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.

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Biology

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction
Palak G. Patel 1,2, Shamini Selvarajah 1,2, Suzanne Boursalie 1,2, Nathan E. How 1,2, Joshua Ejdelman 3, Karl-Philippe Guerard 3, John M. Bartlett 4, Jacques Lapointe 3, Paul C. Park 1, John B. A. Okello 1,2, David M. Berman 1,2
1Department of Pathology & Molecular Medicine, Queen's University, 2Division of Cancer Biology & Genetics, Queen's Cancer Research Institute, Queen's University, 3Department of Surgery, Division of Urology, McGill University, 4Transformative Pathology Program, Ontario Institute for Cancer Research (OICR)

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

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Biochemistry

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification
Melissa Bredow 1, Heather E. Tomalty 1, Virginia K. Walker 1,2,3
1Department of Biology, Queen's University, 2Department of Biomedical and Molecular Sciences, Queen's University, 3School of Environmental Sciences, Queen's University

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.

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Bioengineering

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms
Anna Kornmuller 1, Cody F.C. Brown 2, Claire Yu 3, Lauren E. Flynn 2,4
1Biomedical Engineering Graduate Program, The University of Western Ontario, 2Department of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, The University of Western Ontario, 3Department of Chemical Engineering, Queen's University, 4Department of Chemical & Biochemical Engineering, Faculty of Engineering, The University of Western Ontario

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.

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Behavior

Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice
Karlaina JL Osmon 1, Meera Vyas 1, Evan Woodley 2, Patrick Thompson 2, Jagdeep S Walia 1,2,3
1Centre for Neuroscience Studies, Queen's University, 2Department of Biomedical and Molecular Sciences, Queen's University, 3Medical Genetics/ Department of Pediatrics, Kingston General Hospital, Centre for Neuroscience Studies, and Department of Biomedical and Molecular Sciences, Queen's University

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.

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Biology

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production
Sarah M. Maritan *1,2, Eric Y. Lian *1,2, Lois M. Mulligan 1,2
1Division of Cancer Biology and Genetics, Queen's University Cancer Research Institute, 2Department 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.

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Medicine

Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation
Alison Michels 1, Laura L. Swystun 1, Jeffrey Mewburn 2, Silvia Albánez 1, David Lillicrap 1
1Department of Pathology and Molecular Medicine, Queen's University, 2Department of Medicine, Queen's University

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.

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Neuroscience

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay
Jason C. Campbell 1, Ian D. Chin-Sang 1, William G. Bendena 1,2
1Department of Biology, Queen's University, 2Centre for Neuroscience, Queen's University

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.

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Engineering

A 3D-printed Chamber for Organic Optoelectronic Device Degradation Testing
Emma Mogus 1, Benjamin Torres-Kulik 1, Christopher Gustin 2, Ayse Turak 1
1Department of Engineering Physics, McMaster University, 2Department of Physics, Engineering Physics and Astronomy, Queen's University

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.

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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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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 Rensburg 1, Yawei Hao 1, Xiaolong Yang 1
1Department of Pathology and Molecular Medicine, Queen's University

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.

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Biology

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples
Alison M. Moore 1,2, Lee R. Boudreau 3, Shakeel Virk 3, David P. LeBrun 1,2
1Department of Pathology and Molecular Medicine, Queen's University, 2Division of Cancer Biology and Genetics, Queen's Cancer Research Institute, 3Queen's Laboratory for Molecular Pathology, Department of Pathology and Molecular Medicine, Queen's University

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.

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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 Nishioka 3,5, Yoshitaka Fujimoto 3, Stephen L. Archer 1, Susumu Minamisawa 1,4
1Department of Medicine, Queen's University, 2Department of Biomedical and Molecular Sciences, Queen's University, 3Department of Cell Physiology, The Jikei University School of Medicine, 4Department of Pediatrics, The Jikei University School of Medicine, 5Department of Cardiovascular Surgery, The Jikei University School of Medicine

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.

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Immunology and Infection

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana
Melissa Bredow 1, Irina Sementchoukova 1, Kristen Siegel 1, Jacqueline Monaghan 1
1Department of Biology, Queen's University

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.

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Cancer Research

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research
Zhuo Li *1, Xiao Yang *1, Yilu Zhang 1, Xiaolong Yang 1, Xinxin Cui 1, Yanjuan Zhang 1, Wenfeng Gong 1, Huichen Bai 1, Ning Liu 1, Zhiyu Tang 1, Mingming Guo 1, Kang Li 1, Tong Zhang 1, Lai Wang 1, Xiaomin Song 1
1BeiGene (Beijing) Co., Ltd.

We describe a human peripheral blood mononuclear cell (PBMC) — based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

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Biology

Visually Sexing Loggerhead Shrike (Lanius Ludovicianus) Using Plumage Coloration and Pattern
Gareth Morgan 1, Amy A. Chabot 1,2
1African Lion Safari, 2Department of Biology, Queen's University

We present a protocol to characterize the sex of loggerhead shrike visually based on the coloration and pattern of the sixth primary wing feather.

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Cancer Research

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells
Mulu Geletu *1,2, Victoria Hoskin *1, Blerta Starova *1,3, Maximilian Niit 1,4, Hanad Adan 1, Bruce Elliott 1, Patrick Gunning 2, Leda Raptis 1
1Department of Biomedical and Molecular Sciences and Department of Pathology and Molecular Medicine, Queen's University, Kingston, 2Department of Chemical and Physical Sciences, University of Toronto, Mississauga, 3Department of Laboratory Medicine, Brampton Civic Hospital Campus, William Osler Health System, 4Latner Thoracic Surgery Research Laboratories

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.

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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. McKiel 1, Kimberly A. Woodhouse 1, Lindsay E. Fitzpatrick 1
1Department of Chemical Engineering, Queen's University

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.

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Medicine

Biventricular Assessment of Cardiac Function and Pressure-Volume Loops by Closed-Chest Catheterization in Mice
Francois Potus 1, Ashley Y Martin 1, Brooke Snetsinger 1, Stephen L. Archer 1
1Department of Medicine, Queen's University

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.

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Immunology and Infection

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection
Taylor R. Jamieson 1,2, Joanna Poutou 1,2, Ricardo Marius 1,2, Xiaohong He 1, Reza Rezaei 1,2, Taha Azad 1,2, Carolina S. Ilkow 1,2
1Ottawa Hospital Research Institute, 2Department of Biochemistry, Microbiology and Immunology, University of Ottawa

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

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Immunology and Infection

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter
Reza Rezaei 1,2, Abera Surendran 1,2, Ragunath Singaravelu 1,2, Taylor R. Jamieson 1,2, Parisa Taklifi 3, Joanna Poutou 1,2, Taha Azad 1,2, Carolina S. Ilkow 1,2
1Ottawa Hospital Research Institute, 2Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 3Department of Biotechnology, University of Tehran

The outlined protocol describes the procedure for producing the HiBiT-receptor-binding domain protein complex and its application for fast and sensitive detection of SARS-CoV-2 antibodies.

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