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24 ARTICLES PUBLISHED IN JoVE

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Biology

Culture of myeloid dendritic cells from bone marrow precursors
Jeanette Boudreau 1,2, Sandeep Koshy 2,3, Derek Cummings 2, Yonghong Wan 2
1Medical Sciences Program, McMaster University, 2Centre for Gene Therapeutics, McMaster University, 3Department of Chemical Engineering, University of Waterloo

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

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Biology

A Tactile Automated Passive-Finger Stimulator (TAPS)
Daniel Goldreich 1,2, Michael Wong 2, Ryan M. Peters 2, Ingrid M. Kanics 1
1Department of Occupational Therapy, Duquesne University, 2Department of Psychology, Neuroscience and Behaviour, McMaster University

We describe a computer-controlled device for investigating the sense of touch: the Tactile Automated Passive-finger Stimulator (TAPS). We describe the components of TAPS, and show how TAPS is used to administer a two-interval forced-choice tactile grating orientation test.

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Biology

Primary Human Bronchial Epithelial Cells Grown from Explants
Asma Yaghi 1, Aisha Zaman 1, Myrna Dolovich 1
1Medicine, Faculty of Health Sciences, McMaster University

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

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Immunology and Infection

Detection of Bacteria Using Fluorogenic DNAzymes
Sergio D. Aguirre 1, M. Monsur Ali 1, Pushpinder Kanda 1, Yingfu Li 1,2
1Department of Biochemistry and Biomedical Sciences, McMaster University , 2Department of Chemistry and Chemical Biology, McMaster University

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

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Medicine

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice
Jane A. E. Batt 1, James Ralph Bain 2
1Keenan Research Centre of the LiKaShing Knowledge Institute, St Michaels Hospital, 2Department of Surgery, McMaster University

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice.  

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Medicine

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus
Daniel E. Venegas-Pino 1, Nicole Banko 1, Mohammed I. Khan 2, Yuanyuan Shi 2, Geoff H. Werstuck 1,2
1Department of Biochemistry and Biomedical Sciences, McMaster University, 2Thrombosis and Atherosclerosis Research Institute, McMaster University

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

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Behavior

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)
Boris Sakic 1, Marcella P. A. Cooper 2, Sarah E. Taylor 2, Milica Stojanovic 2, Bosa Zagorac 2, Minesh Kapadia 3
1Department of Psychiatry and Behavioral Neurosciences, McMaster University, 2Department of Psychology, Neuroscience & Behaviour, McMaster University, 3Neuroscience Program, McMaster University

Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis.

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Behavior

Design and Implementation of an fMRI Study Examining Thought Suppression in Young Women with, and At-risk, for Depression
Caitlin L. Carew 1, Erica L. Tatham 2, Andrea M. Milne 1, Glenda M. MacQueen 3, Geoffrey B.C. Hall 4
1Department of Psychiatry and Behavioural Neurosciences, McMaster University, 2McMaster Integrative Neuroscience Discovery and Study, McMaster University, 3Department of Psychiatry, University of Calgary, 4Department of Psychology, Neuroscience & Behaviour, McMaster University

We aim to identify the neural correlates underlying sustained and transient thought suppression, and thought re-emergence in controls, at-risk and depressed individuals. Activation was greatest for controls compared to the at-risk and the depressed group in the dorsolateral prefrontal cortex during thought suppression and anterior cingulate cortex during thought re-emergence.

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Medicine

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses
Breanne Cuddington 1, Meghan Verschoor 1, Karen Mossman 1
1Department of Pathology and Molecular Medicine, McMaster Immunology Research Centre, Institute for Infectious Disease Research, McMaster University

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

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Behavior

Combining Multiple Data Acquisition Systems to Study Corticospinal Output and Multi-segment Biomechanics
Michael J. Asmussen 1, Aaron Z. Bailey 1, Peter J. Keir 1, Jim Potvin 1, Tim Bergel 2, Aimee J. Nelson 1
1Department of Kinesiology, McMaster University, 22Els Espots

The use of transcranial magnetic stimulation (TMS) to study human motor control requires the integration of data acquisition systems to control TMS delivery and simultaneously record human behavior. The present manuscript provides a detailed methodology for integrating data acquisition systems for the purpose of investigating human movement via TMS.

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Bioengineering

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning
Daryl Sivakumaran 1, Emilia Bakaic 1, Scott B. Campbell 1, Fei Xu 1, Eva Mueller 1, Todd Hoare 1
1Department of Chemical Engineering, McMaster University

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

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Biology

Colorimetric Detection of Bacteria Using Litmus Test
Kha Tram 1, Sepehr Manochehry 1, Qian Feng 2, Dingran Chang 1, Bruno J. Salena 3, Yingfu Li 1
1Department of Biochemistry and Biomedical Sciences, McMaster University, 2Department of Chemistry and Chemical Biology, McMaster University, 3Department of Medicine, McMaster University

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

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Neuroscience

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors
Kelsey Lee *1, Ou Chong Saetern *1, Andrew Nguyen 1, Leonardo Rodriguez 1, Birgitt Schüle 1
1Parkinson’s Institute and Clinical Center

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

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Chemistry

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry
Holly A. Bilton *1, Zainab Ahmad *1, Nancy Janzen 1, Shannon Czorny 1, John F. Valliant 1
1Department of Chemistry and Chemical Biology, McMaster University

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

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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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Biology

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1
Lucas Le Nagard 1, Viviana Morillo-López 2, Cecile Fradin 1, Dennis A. Bazylinski 2
1Department of Physics & Astronomy, McMaster University, 2School of Life Sciences, University of Nevada Las Vegas

We present a procedure for growing several strains of Magnetospirillum in two different types of growth media. Magnetospirillum gryphiswaldense strain MSR-1 is grown in both liquid and O2 concentration gradient semi-solid media while M. magneticum strain AMB-1 and M. magnetotacticum strain MS-1 are grown in liquid medium.

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Chemistry

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry
Meera Shanmuganathan 1, Sabrina Macklai 1, Clara Barrenas Cárdenas 1, Zachary Kroezen 1, Marcus Kim 2, William Zizek 3, Howard Lee 3, Philip Britz-McKibbin 1
1Department of Chemistry and Chemical Biology, McMaster University, 2Agilent Technologies Inc., 3Seroclinix Corporation

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

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Immunology and Infection

Antibiotic Dereplication Using the Antibiotic Resistance Platform
Haley L. Zubyk 1, Georgina Cox 2, Gerard D. Wright 1
1Department of Biochemistry and Biomedical Sciences, M.G. DeGroote Institute for Infectious Disease Research, McMaster University, 2Department of Molecular and Cellular Biology, University of Guelph

We describe a platform that utilizes a library of isogenic antibiotic resistant Escherichia coli for the dereplication of antibiotics. The identity of an antibiotic produced by bacteria or fungi can be deduced by the growth of E. coli expressing its respective resistance gene. This platform is economically effective and time-efficient.

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Cancer Research

Functional Analysis of Tumor-Infiltrating Myeloid Cells by Flow Cytometry and Adoptive Transfer
Andrew Nguyen *1, Omar Salem *1, Yonghong Wan 1
1Department of Pathology and Molecular Medicine, McMaster University

This protocol provides reliable methods of solid tumor dissociation and myeloid cell isolation in murine intradermal or subcutaneous tumor models. Flow cytometry allows for phenotypic characterization of heterogeneous myeloid populations within the tumor microenvironment and sorting will demonstrate their functionality in the context of adoptive transfer.

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Neuroscience

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis
Darren M. O'Hara 1,2, Minesh Kapadia 1,2, Susan Ping 1,2, Suneil K. Kalia 1,2,3, Lorraine V. Kalia 1,2,4,5,6
1Krembil Research Institute, Toronto Western Hospital, University Health Network, 2Department of Laboratory Medicine and Pathobiology, University of Toronto, 3Department of Surgery, Division of Neurosurgery, University of Toronto, 4Department of Medicine, Division of Neurology, University of Toronto, 5Department of Medicine, Division of Neurology, Edmond J. Safra Program in Parkinson's Disease and the Morton and Gloria Shulman Movement Disorders Clinic, Toronto Western Hospital, University Health Network, 6Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

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Biology

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure
Himeshi Samarasinghe 1, Gregory Korfanty 1, Jianping Xu 1
1Department of Biology, McMaster University

This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments.

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Biochemistry

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice
Liza-Anastasia DiCecco *1,2, Samantha Berry *1, G. M. Jonaid *1,3, Maria J. Solares 1,4, Liam Kaylor 1,4, Jennifer L. Gray 5, Carol Bator 6, William J. Dearnaley 1, Michael Spilman 7, Madeline J. Dressel-Dukes 8, Kathryn Grandfield 2, Sarah M. McDonald Esstman 9, Deborah F. Kelly 1,5,6
1Department of Biomedical Engineering, Pennsylvania State University, 2Department of Materials Science and Engineering, McMaster University, 3Bioinformatics and Genomics Graduate Program, Huck Institutes of the Life Sciences, Pennsylvania State University, 4Molecular, Cellular, and Integrative Biosciences Graduate Program, Huck Institutes of the Life Sciences, Pennsylvania State University, 5Materials Research Institute, Pennsylvania State University, 6Huck Institutes of the Life Sciences, Pennsylvania State University, 7Applications team, Direct Electron, 8Application Scientist, Protochips, Inc., 9Department of Biology, Wake Forest University

Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.

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Engineering

A Teleoperated Robotic System-Assisted Percutaneous Transiliac-Transsacral Screw Fixation Technique
Binbin Liu 1, Fei Xu 1, Yang Liu 1, Tianqi Wang 2, Yajing Cao 2, Zehang Zheng 1, Hanqing Xu 1, Chuang Huang 1, Zhengqiang Luo 1
1Department of Orthopedics, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, 2Wuhan United Imaging Healthcare Surgical Technology Co. Ltd.

Teleoperated robotic system-assisted percutaneous transiliac-transsacral screw fixation is a feasible technique. Screw channels can be implemented with high accuracy owing to the excellent freedom of movement and stability of the robotic arms.

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