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168 ARTICLES PUBLISHED IN JoVE

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Biology

Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging
Melissa Paulite 1, Zahra Fakhraai 2, Boris B. Akhremitchev 3, Kerstin Mueller 1, Gilbert C. Walker 1
1Department of Chemistry, University of Toronto, 2Department of Chemistry, University of Wisconsin, 3Department of Chemistry, Duke University

The assembly of a nearfield infrared microscope for imaging protein aggregates is described.

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Biology

Digital Microfluidics for Automated Proteomic Processing
Mais J. Jebrail 1, Vivienne N. Luk 1,2, Steve C. C. Shih 2,3, Ryan Fobel 2,3, Alphonsus H. C. Ng 2,3, Hao Yang 1, Sergio L. S. Freire 1, Aaron R. Wheeler 1,2,3
1Department of Chemistry, University of Toronto, 2Donnelly Centre for Cellular and Biomolecular Research, 3Institute for Biomaterials and Biomedical Engineering, University of Toronto

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

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Biology

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions
Jamie Snider 1,2,3, Saranya Kittanakom 1,2,3, Jasna Curak 1,2,3, Igor Stagljar 1,2,3
1Department of Biochemistry, University of Toronto, 2Department of Molecular Genetics, University of Toronto, 3Terrence Donnelly Centre for Cellular and Biomolecular Research (CCBR), University of Toronto

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

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Immunology and Infection

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry
Christina Farr 1, Stuart Berger 1,2
1Immunology, University of Toronto, 2Arthritis and Immune Disorder Research Centre, University Health Network (UHN)

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

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Biology

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase
Usheer Kanjee 1, Walid A. Houry 1
1Department of Biochemistry, University of Toronto

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

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Neuroscience

Eye Movement Monitoring of Memory
Jennifer D. Ryan 1,2,3, Lily Riggs 1,2, Douglas A. McQuiggan 1
1Rotman Research Institute, 2Department of Psychology, University of Toronto, 3Department of Psychiatry, University of Toronto

Eye movement monitoring (or eye tracking) reveals where in space the eyes linger, when and for how long. Here, we demonstrate how eye tracking can be used to investigate the integrity of memory in multiple participant populations, without requiring verbal, or otherwise explicit, reports.

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Immunology and Infection

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells
Ahmad Zaheen 1, Alberto Martin 1
1Department of Immunology, University of Toronto

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

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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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Biology

Isolation of Retinal Stem Cells from the Mouse Eye
Brenda L.K. Coles 1, Derek van der Kooy 1
1Molecular Genetics, University of Toronto

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

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Bioengineering

Microfabricated Platforms for Mechanically Dynamic Cell Culture
Christopher Moraes 1,2, Yu Sun 1,2, Craig A. Simmons 1,2,3
1Department of Mechanical and Industrial Engineering, University of Toronto, 2Institute of Biomaterials and Biomedical Engineering, University of Toronto, 3Faculty of Dentistry, University of Toronto

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

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Medicine

An Investigation of the Effects of Sports-related Concussion in Youth Using Functional Magnetic Resonance Imaging and the Head Impact Telemetry System
Michelle Keightley 1,2,3,4,5, Stephanie Green 1, Nick Reed 1, Sabrina Agnihotri 1, Amy Wilkinson 3, Nancy Lobaugh 6,7
1Graduate Department of Rehabilitation Science, University of Toronto, 2Occupational Science and Occupational Therapy, University of Toronto, 3Department of Psychology, University of Toronto, 4Bloorview Kids Rehab, 5Toronto Rehab, 6Cognitive Neurology, Sunnybrook Health Sciences Centre, 7Faculty of Medicine, University of Toronto

This article provides an overview of a multi-modal approach to mild traumatic brain injury diagnosis and recovery in youth. This approach combines neuropsychological testing with functional magnetic resonance imaging and the Head Impact Telemetry System to monitor the relationship between head impacts and brain activity during cognitive testing.

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Neuroscience

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System
Mark M. Magharious *1, Philippe M. D'Onofrio *1, Paulo D. Koeberle 1
1Department of Surgery, University of Toronto

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

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Biology

Dissection of Oenocytes from Adult Drosophila melanogaster
Joshua J. Krupp 1, Joel D. Levine 1
1Department of Biology, University of Toronto

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

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Neuroscience

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS
Philippe M. D'Onofrio *1, Mark M. Magharious *1, Paulo D. Koeberle 1
1Department of Surgery, University of Toronto

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

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Neuroscience

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises
Brittany Baierlein *1, Alison L. Thurow *1, Harold L. Atwood *2, Robin L. Cooper *1
1Department of Biology, University of Kentucky, 2Department of Physiology, University of Toronto

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

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Education

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises
Martha M. Robinson 1, Jonathan M. Martin 1, Harold L. Atwood 2, Robin L. Cooper 1
1Department of Biology, University of Kentucky, 2Department of Physiology, University of Toronto

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

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Biology

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection
Serge Gueroussov 1, Stefan P. Tarnawsky 1, Xianying A. Cui 1, Kohila Mahadevan 1, Alexander F. Palazzo 1
1Department of Biochemistry, University of Toronto

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

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Medicine

A Protocol for Comprehensive Assessment of Bulbar Dysfunction in Amyotrophic Lateral Sclerosis (ALS)
Yana Yunusova 1,2, Jordan R. Green 3, Jun Wang 3, Gary Pattee 4, Lorne Zinman 2,5
1Department of Speech-Language Pathology, University of Toronto, 2ALS/ MN Clinic, Sunnybrook Health Science Centre, 3Department of Special Education and Communication Disorders, University of Nebraska-Lincoln, 4Department of Neurology, Munroe-Meyer Institute, University of Nebraska Medical Center, 5Department of Neurology, University of Toronto

Objective assessments of the physiological mechanisms that support speech are needed to monitor disease onset and progression in persons with ALS and to quantify treatment effects in clinical trials. In this video, we present a comprehensive, instrumentation-based protocol for quantifying speech motor performance in clinical populations.

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Neuroscience

Assessment of Social Interaction Behaviors
Oksana Kaidanovich-Beilin 1, Tatiana Lipina 1, Igor Vukobradovic 2, John Roder 1,3,4,5, James R. Woodgett 1,3
1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 2Toronto Centre for Phenogenomics, Mount Sinai Hospital, 3Department of Medical Biophysics, University of Toronto, 4Department of Psychology, University of Toronto, 5Department of Psychiatry, University of Toronto

Here we describe a detailed protocol for examination of sociability in mice by using Crawley's sociability and preference for social novelty test. We describe the advantages and possible applications for this procedure, including critical details important for correct interpretation of the results.

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Neuroscience

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging
Jun Dazai *1, Shoshana Spring *1, Lindsay S. Cahill 1, R. Mark Henkelman 1,2
1Mouse Imaging Centre, Hospital for Sick Children, 2Department of Medical Biophysics and Medical Imaging, University of Toronto

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

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Neuroscience

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions
Vera Brümmer 1, Stefan Schneider 1, Tobias Vogt 1, Heiko Strüder 1, Heather Carnahan 2, Christopher D. Askew 3, Roland Csuhaj 4
1Institute of Movement and Neurosciences, German Sport University Cologne, 2Deptartment of Surgical Skills, University of Toronto, 3School of Human Movement Studies, Institute of Health and Biomedical Innovation, Queensland University of Technology, 4Brain Products GmbH, Scientific Support, Gilching, Germany

The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

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Biology

Competitive Genomic Screens of Barcoded Yeast Libraries
Andrew M. Smith *1,2, Tanja Durbic *2,3, Julia Oh *4, Malene Urbanus 1,2, Michael Proctor 5, Lawrence E. Heisler 2,3, Guri Giaever 2,6, Corey Nislow 1,2,3
1Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, 2Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 3Donnelly Sequencing Centre, University of Toronto, 4Genetics and Molecular Biology Branch, National Human Genome Research Institute, NIH, 5Stanford Genome Technology Center, Stanford School of Medicine, Stanford University , 6Department of Pharmaceutical Sciences, University of Toronto

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

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Medicine

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model
Meaghan A. O'Reilly 1, Adam C. Waspe 1,2, Rajiv Chopra 1,2, Kullervo Hynynen 1,3
1Imaging Research, Sunnybrook Research Institute, 2Department of Medical Biophysics, University of Toronto, 3Department of Medical Biophysics, and Institute of Biomaterials & Biomedical Engineering (IBBME), University of Toronto

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

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Medicine

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging
Jill J. Weyers 1, Dara D. Carlson 1, Charles E. Murry 1,2, Stephen M. Schwartz 1, William M. Mahoney, Jr. 1
1Department of Pathology, Center for Cardiovascular Biology, and Institute for Stem Cell and Regenerative Medicine, University of Washington, 2Departments of Bioengineering and Medicine/Cardiology, University of Washington

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (μCT) imaging.

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Medicine

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)
Aida Gordanpour 1, Robert K. Nam 2, Linda Sugar 3, Stephanie Bacopulos 1, Arun Seth 1,3,4
1Department of Laboratory Medicine & Pathobiology, University of Toronto, 2Division of Urology, Sunnybrook Health Sciences Centre, Toronto, Canada, 3Department of Anatomic Pathology, Sunnybrook Health Sciences Centre, Toronto, Canada, 4Biological Sciences, Sunnybrook Research Institute

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

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Biology

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates
Faiza Waheed 1,2, Pamela Speight 1,2, Qinghong Dan 1,2, Rafael Garcia-Mata 3, Katalin Szaszi 1,2
1Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital , 2Department of Surgery, University of Toronto, 3Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill

The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.

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Immunology and Infection

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation
Rahul Kushwah 1, Jim Hu 2
1Stem Cell and Cancer Research Institute, McMaster University, Hamilton, 2Physiology and Experimental Medicine Research Program, Hospital for Sick Children, University of Toronto

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

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Biology

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells
Halil Aydin *1, Farshad C. Azimi *1, Jonathan D. Cook *1, Jeffrey E. Lee 1
1Department of Laboratory Medicine and Pathobiology, University of Toronto

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

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Biology

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
Alla Gagarinova *1, Mohan Babu *2,3, Jack Greenblatt 1,2, Andrew Emili 1,2
1Department of Molecular Genetics, University of Toronto, 2Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 3Department of Biochemistry, Research and Innovation Centre, University of Regina

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

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Biology

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry
Mohan Babu 1,2, Olga Kagan 1, Hongbo Guo 1, Jack Greenblatt 1,3, Andrew Emili 1,3
1Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 2Deparment of Biochemistry, Research and Innovation Centre, University of Regina, 3Department of Medical Genetics and Microbiology, University of Toronto

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

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Biology

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions
Yo Suzuki 1, Jason Stam 1, Mark Novotny 2, Nozomu Yachie 3, Roger S. Lasken 2, Frederick P. Roth 3,4
1Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, 2Department of Microbial and Environmental Genomics, J. Craig Venter Institute, 3Donnelly Centre & Department of Molecular Genetics, University of Toronto, 4Lunenfeld Research Institute, Mt Sinai Hospital

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

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Neuroscience

C. elegans Tracking and Behavioral Measurement
Jirapat Likitlersuang 1, Greg Stephens 2,3, Konstantine Palanski 1, William S. Ryu 1,4
1Donnelly Centre, University of Toronto, 2Department of Physics and Astronomy, Vrije Universiteit, 3Okinawa Institute of Science and Technology, 4Department of Physics, University of Toronto

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

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Neuroscience

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field
Robart Babona-Pilipos 1, Milos R. Popovic 2, Cindi M. Morshead 3
1Institute for Biomaterials and Biomedical Engineering, University of Toronto, 2Lyndhurst Centre, Toronto Rehabilitation Institute, 3Department of Surgery, University of Toronto

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

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Engineering

Compact Quantum Dots for Single-molecule Imaging
Andrew M. Smith 1, Shuming Nie 1,2
1Department of Biomedical Engineering, Emory University, 2Department of Chemistry, Georgia Institute of Technology

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

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Bioengineering

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen
Richard W. Loo 1,2, Jane Betty Goh 1,2, Calvin C.H. Cheng 1, Ning Su 1, M. Cynthia Goh 1,2
1Department of Chemistry, University of Toronto, 2Institute for Optical Sciences, University of Toronto

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

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Engineering

Bringing the Visible Universe into Focus with Robo-AO
Christoph Baranec 1,2, Reed Riddle 1, Nicholas M. Law 3, A.N. Ramaprakash 4, Shriharsh P. Tendulkar 2, Khanh Bui 1, Mahesh P. Burse 4, Pravin Chordia 4, Hillol K. Das 4, Jack T.C. Davis 1, Richard G. Dekany 1, Mansi M. Kasliwal 5, Shrinivas R. Kulkarni 1,2, Timothy D. Morton 2, Eran O. Ofek 6, Sujit Punnadi 4
1Caltech Optical Observatories, California Institute of Technology, 2Department of Astronomy, California Institute of Technology, 3Dunlap Institute for Astronomy and Astrophysics, University of Toronto, 4Inter-University Centre for Astronomy & Astrophysics, 5Observatories of the Carnegie Institution for Science, 6Benoziyo Center for Astrophysics, Weizmann Institute of Science

Light from astronomical objects must travel through the earth's turbulent atmosphere before it can be imaged by ground-based telescopes. To enable direct imaging at maximum theoretical angular resolution, advanced techniques such as those employed by the Robo-AO adaptive-optics system must be used.

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Biology

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells
Xianying A. Cui 1, Alexander F. Palazzo 1
1Department of Biochemistry, University of Toronto

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

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Medicine

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics
Kelly Burrell 1, Sameer Agnihotri 1, Michael Leung 2, Ralph DaCosta 2, Richard Hill 2, Gelareh Zadeh 3
1Brain Tumor Research Centre, Hospital for Sick Children, Toronto Medical Discovery Tower, 2Ontario Cancer Institute, Princess Margaret Hospital, 3Neurosurgery, Toronto Western Hospital

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

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Chemistry

Template Directed Synthesis of Plasmonic Gold Nanotubes with Tunable IR Absorbance
Colin R. Bridges 1, Tyler B. Schon 1, Paul M. DiCarmine 1, Dwight S. Seferos 1
1Department of Chemistry, University of Toronto

Solution-suspendable gold nanotubes with controlled dimensions can be synthesized by electrochemical deposition in porous anodic aluminum oxide (AAO) membranes using a hydrophobic polymer core. Gold nanotubes and nanotube arrays hold promise for applications in plasmonic biosensing, surface-enhanced Raman spectroscopy, photo-thermal heating, ionic and molecular transport, microfluidics, catalysis and electrochemical sensing.

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Bioengineering

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles
Cheng S. Jin 1,2,3, Jonathan F. Lovell 4, Gang Zheng 1,2,3
1Department of Pharmaceutical Sciences, University of Toronto, 2The Institute of Biomaterials and Biomedical Engineering, University of Toronto, 3Ontario Cancer Institute, Campbell Family Institute For Cancer Research and Techna Institute, 4Department of Biomedical Engineering, University at Buffalo, The State University of New York

We developed novel intrinsic multifunctional nanovesicles called porphysomes, which have structure-dependent fluorescence self-quenching and unique photothermal properties, thus functioning as potent photothermal therapy agents. We formulated porphysomes using high pressure extrusion and investigated their photothermal therapy efficacy in a xenograft tumor model.

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Bioengineering

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments
Anupama Sinha 1, Mais J. Jebrail 2, Hanyoup Kim 2,3, Kamlesh D. Patel 4, Steven S. Branda 2
1Department of Systems Biology, Sandia National Laboratories, 2Department of Biotechnology and Bioengineering, Sandia National Laboratories, 3Canon U.S. Life Sciences, 4Department of Advanced Systems Engineering and Deployment, Sandia National Laboratories

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

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Medicine

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly
Joel Ramirez 1, Christopher J.M. Scott 1, Alicia A. McNeely 1, Courtney Berezuk 1, Fuqiang Gao 1, Gregory M. Szilagyi 1,2, Sandra E. Black 1,2
1LC Campbell Cognitive Neurology Research Unit, Heart & Stroke Foundation Canadian Partnership for Stroke Recovery, Brain Sciences Research Program, Sunnybrook Health Sciences Centre, 2Department of Medicine (Neurology), Institute of Medical Science, University of Toronto

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

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Bioengineering

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage
Spencer Bell 1, Elnaz Ajami 1, John E. Davies 1
1Institute of Biomaterials and Biomedical Engineering, University of Toronto

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

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Biology

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands
Rushin Shojaii 1, Stephanie Bacopulos 2,3, Wenyi Yang 2,3, Tigran Karavardanyan 4, Demetri Spyropoulos 5, Afshin Raouf 6, Anne Martel 1,4, Arun Seth 2,3
1Department of Medical Biophysics, University of Toronto, 2Platform Biological Sciences, Sunnybrook Research Institute, 3Department of Laboratory Medicine and Pathobiology, University of Toronto, 4Physical Sciences, Sunnybrook Research Institute, 5Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 6Manitoba Institute of Cell Biology, University of Manitoba

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

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Immunology and Infection

Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries
Shane Miersch 1,2, Zhijian Li 1,2, Rachel Hanna 1,2, Megan E. McLaughlin 1,2, Michael Hornsby 1,3, Tet Matsuguchi 1,3, Marcin Paduch 1,4, Annika Sääf 1,4, Jim Wells 1,3, Shohei Koide 1,4, Anthony Kossiakoff 1,4, Sachdev S. Sidhu 1,2
1The Recombinant Antibody Network, 2The Banting and Best Department of Medical Research, University of Toronto, 3Antibiome Center, University of California, San Francisco at Mission Bay, 4Department of Biochemistry and Molecular Biology, The University of Chicago

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

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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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Neuroscience

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging
Julie Winterburn 1,2, Jens C. Pruessner 3, Chavez Sofia 4,5, Mark M. Schira 6,7, Nancy J. Lobaugh 4,8, Aristotle N. Voineskos 5,9, M. Mallar Chakravarty 1,2
1Institute of Biomaterials and Biomedical Engineering, University of Toronto, 2Computational Brain Anatomy Laboratory, Douglas Institute, McGill University, 3McGill Centre for Studies in Aging, McGill University, 4MRI Unit, Research Imaging Centre, Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, 5Department of Psychiatry, University of Toronto, 6School of Psychology, University of Wollongong, 7Neuroscience Research Australia, 8Department of Medicine, University of Toronto, 9Kimel Family Translational Imaging Genetics Research Laboratory, Research Imaging Centre, Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

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Environment

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers
Devrim Coskun 1, Dev T. Britto 1, Ahmed M. Hamam 1, Herbert J. Kronzucker 1
1Department of Biological Sciences, University of Toronto

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

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Medicine

A Multi-Modal Approach to Assessing Recovery in Youth Athletes Following Concussion
Nick Reed 1,2,3, James Murphy 1, Talia Dick 1, Katie Mah 3, Melissa Paniccia 3, Lee Verweel 3, Danielle Dobney 3, Michelle Keightley 1,2,3
1Bloorview Research Institute, Holland Bloorview Kids Rehabilitation Hospital, 2Department of Occupational Science and Occupational Therapy, University of Toronto, 3Graduate Department of Rehabilitation Science, University of Toronto

This article provides an overview of a multi-modal approach to assessing recovery following concussion in youth athletes. The described protocol uses pre- and post-concussion assessment of performance across a wide variety of domains and can inform the development of improved concussion rehabilitation protocols specific to the youth sport community.

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Neuroscience

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient
Marie Kristel Bermejo *1, Marija Milenkovic *1, Ali Salahpour 1, Amy J. Ramsey 1
1Department of Pharmacology and Toxicology, University of Toronto

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

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JoVE Core

Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics
Sergio L. S. Freire 1, Nathaniel Thorne 1, Michael Wutkowski 1, Selina Dao 1
1Department of Mathematics, Physics and Statistics, University of the Sciences

The protocol for fabrication and operation of field dewetting devices (Field-DW) is described, as well as the preliminary studies of the effects of electric fields on droplet contents.

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Neuroscience

Long-term Time Lapse Imaging of Mouse Cochlear Explants
Joanna F. Mulvaney 1, Alain Dabdoub 1,2,3
1Biological Sciences Platform, Sunnybrook Research Institute, 2Department of Otolaryngology - Head and Neck Surgery, University of Toronto, 3Department of Laboratory Medicine and Pathobiology, University of Toronto

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

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Medicine

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury
Klaus Zweckberger 1, Yang Liu 1, Jian Wang 1, Nicole Forgione 1, Michael G. Fehlings 1,2,3
1Department of Genetics and Development, Toronto Western Research Institute and Spinal Program, University Health Network, Krembil Neuroscience Centre, 2Department of Surgery, University of Toronto, 3Institute of Medical Sciences, University of Toronto

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

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Immunology and Infection

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells
Martina Kučerová-Levisohn 1, Jordana Lovett 1, Armin Lahiji 1, Roxanne Holmes 2, Juan Carlos Zúñiga-Pflücker 2, Benjamin D. Ortiz 1
1Department of Biological Sciences, Hunter College and Graduate Center, City University of New York, 2Sunnybrook Research Institute, Department of Immunology, University of Toronto

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

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Neuroscience

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun
Jason Arsenault 1,2, Andras Nagy 1, Jeffrey T. Henderson 1, John A. O'Brien 2
1Leslie Dan Faculty of Pharmacy, University of Toronto, 2MRC-Laboratory of Molecular Biology, Cambridge, UK

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

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Immunology and Infection

Electroporation of Functional Bacterial Effectors into Mammalian Cells
Ryan L. Sontag 1, Cosmin Mihai 2, Galya Orr 2, Alexei Savchenko 3, Tatiana Skarina 3, Hong Cui 3, John R. Cort 1, Joshua N. Adkins 1, Roslyn N. Brown 4
1Biological Sciences Division, Pacific Northwest National Laboratory, 2Environmental Molecular Science Laboratory, Pacific Northwest National Laboratory, 3Structural Proteomics Group, Ontario Center for Structural Proteomics, University of Toronto, 4Center for Bioproducts and Bioenergy, Washington State University

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

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Developmental Biology

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis
Adam J. Mosa 1, Sabrina Wang 2,3, Yao Fang Tan 1, J. Martin Wojtowicz 1
1Department of Physiology, University of Toronto, 2Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, 3Department of Education and Research, Taipei City Hospital

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

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Biology

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein
Paul D. W. Eckford 1, Canhui Li 1, Christine E. Bear 1,2,3
1Programme in Molecular Structure and Function, Hospital for Sick Children, 2Department of Biochemistry, University of Toronto, 3Department of Physiology, University of Toronto

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

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Developmental Biology

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound
Janet M. Denbeigh 1,2, Brian A. Nixon 1,2, Mira C. Puri 1,2,3, F. Stuart Foster 1,2
1Department of Medical Biophysics, University of Toronto, 2Sunnybrook Research Institute, 3Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

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Medicine

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury
Arnaud Dubory 1,2, Elisabeth Laemmel 1, Anna Badner 3, Jacques Duranteau 1,4, Eric Vicaut 1, Charles Court 2, Marc Soubeyrand 1,2
1Laboratoire d'étude de la microcirculation, Faculté de Médecine Paris Diderot Paris VII, U942, 2Department of orthopaedic surgery, Bicetre Universitary Hospital, Public Assistance of Paris Hospital, 3Institute of Medical Science, Faculty of Medicine, University of Toronto, 4Department of Intensive care and Anesthesiology, Bicetre Universitary Hospital, Public Assistance of Paris Hospital

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

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Behavior

Testing Sensory and Multisensory Function in Children with Autism Spectrum Disorder
Sarah H. Baum 1, Ryan A. Stevenson 2, Mark T. Wallace 3
1Vanderbilt Brain Institute, Vanderbilt University Medical Center, 2Department of Psychology, University of Toronto, 3Department of Hearing and Speech Sciences, Vanderbilt University

We describe how to implement a battery of behavioral tasks to examine the processing and integration of sensory stimuli in children with ASD. The goal is to characterize individual differences in temporal processing of simple auditory and visual stimuli and relate these to higher order perceptual skills like speech perception.

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JoVE Journal

Metal-silicate Partitioning at High Pressure and Temperature: Experimental Methods and a Protocol to Suppress Highly Siderophile Element Inclusions
Neil R. Bennett 1,2, James M. Brenan 1, Yingwei Fei 2
1Department of Earth Science, University of Toronto, 2Geophysical Laboratory, Carnegie Institution of Washington

We present a procedure to determine the metal-silicate partitioning of siderophile elements, emphasizing techniques that suppress the formation of metal inclusions in experiments for the noble metals. The results of these experiments are used to demonstrate the effect of core-formation on the highly siderophile element composition of the mantle.

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Bioengineering

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance
Yannan N. Dou 1, Robert A. Weersink 2,5, Warren D. Foltz 5,6, Jinzi Zheng 5,6, Naz Chaudary 7, David A. Jaffray 2,3,4,5,6, Christine Allen 1
1Leslie Dan Faculty of Pharmacy, University of Toronto, 2Department of Radiation Oncology, University of Toronto, 3Medical Biophysics, University of Toronto, 4Institute of Biomaterials & Biomedical Engineering, University of Toronto, 5Techna Institute and Radiation Medicine Program, Princess Margaret Cancer Center, University Health Network, 6STTARR Innovation Center, University Health Network, 7Ontario Cancer Institute, University Health Network

AN MRI-compatible custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

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Medicine

MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder
Katharine Dunlop 1, Pauline Gaprielian 6, Daniel Blumberger 5,7, Zafiris J. Daskalakis 5,7, Sidney H. Kennedy 2,3,5, Peter Giacobbe 2,3,5, Jonathan Downar 2,3,4,5
1Institute of Medical Sciences, University of Toronto, 2MRI-Guided rTMS Clinic, University Health Network, 3Department of Psychiatry, University Health Network, 4Toronto Western Research Institute, University Health Network, 5Department of Psychiatry, University of Toronto, 6Faculty of Arts and Science, University of Toronto, 7Temerty Centre for Therapeutic Brain Intervention, Centre for Addiction and Mental Health

Here we outline the procedure for MRI-guided repetitive transcranial magnetic stimulation to the dorsomedial prefrontal cortex as an experimental treatment for major depressive disorder.

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Biology

Identification of Kinase-substrate Pairs Using High Throughput Screening
Courtney Reeks 1, Robert A. Screaton 2,3
1Children's Hospital of Eastern Ontario Research Institute, 2Sunnybrook Research Institute, University of Toronto, 3Department of Biochemistry, University of Toronto

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

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Medicine

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies
Emily J. Chadwick 1, David P. Yang 1, Mariella G. Filbin 2, Emanuele Mazzola 3, Yu Sun 1, Oded Behar 1,4, Maria F. Pazyra-Murphy 1, Liliana Goumnerova 5, Keith L. Ligon 6, Charles D. Stiles 1, Rosalind A. Segal 1
1Department of Cancer Biology, Dana-Farber Cancer Institute, 2Department of Pediatrics, Children's Hospital, 3Department of Biostatistics & Computational Biology, Dana-Farber Cancer Institute, 4Department of Developmental Biology and Cancer Research, Hebrew University of Jerusalem, 5Department of Neurosurgery, Children's Hospital, 6Center for Molecular Oncologic Pathology, Department of Medical Oncology, Dana-Farber Cancer Institute

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice.  

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Neuroscience

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem
Larissa McKetton 1, Joy Williams 2, Joseph D. Viviano 1, Yeni H. Yücel 3, Neeru Gupta 3, Keith A. Schneider 1
1Department of Biology and Centre for Vision Research, York University, 2York MRI Facility, York University, 3Department of Ophthalmology & Vision Sciences, Laboratory Medicine & Pathobiology, University of Toronto

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

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Developmental Biology

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells
Sakthi D. Moorthy 1, Jennifer A. Mitchell 1
1Department of Cell and Systems Biology, University of Toronto

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

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Medicine

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model
Lee Chin 1,2, Elina Korpela 3, Anthony Kim 1, Darren Yohan 2, Carolyn Niu 4, Brian C. Wilson 3, Stanley K. Liu 1,3
1Department of Radiation Oncology, University of Toronto, 2Department of Physics, Ryerson University, 3Department of Medical Biophysics, University of Toronto, 4Ontario Cancer Institute / Campbell Family Institute for Cancer Research

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

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Neuroscience

Foraging Path-length Protocol for Drosophila melanogaster Larvae
Ina Anreiter 1, Oscar E. Vasquez 1, Aaron M. Allen 2, Marla B. Sokolowski 1,3
1Department of Ecology and Evolutionary Biology, University of Toronto, 2Department of Cell and Systems Biology, University of Toronto, 3Child and Brain Development Program, Canadian Institute for Advanced Research

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

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Immunology and Infection

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow
Yonghong Chen *1, Azusa Maeda *1,2, Jiachuan Bu 1, Ralph DaCosta 1,2,3
1Princess Margaret Cancer Centre, 2Department of Medical Biophysics, University of Toronto, 3Techna Institute, University Health Network

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

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Bioengineering

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli
Matthew J. Leineweber 1, Sam Shi 2, Jan Andrysek 1,2
1Bloorview Research Institute, Holland Bloorview Kids Rehabilitation Hospital, 2Institute of Biomaterials and Biomedical Engineering, University of Toronto

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant's volitional response for various combinations of stimulation location and frequency.

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Medicine

Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors
Shawn Stapleton 1,2,3, Daniel Mirmilshteyn 2, Jinzi Zheng 3,4, Christine Allen 2,4,5, David A. Jaffray 1,2,3,4,5,6
1Department of Medical Biophysics, University of Toronto, 2Leslie Dan Faculty of Pharmacy, University of Toronto, 3STTARR Innovation Centre, Princess Margaret Cancer Centre, 4Institute of Biomaterials and Biomedical Engineering, University of Toronto, 5Techna Institute, University Health Network, 6Radiation Medicine Program, Princess Margaret Cancer Centre

The heterogeneous intra-tumoral accumulation of liposomes has been linked to an abnormal tumor microenvironment. Herein methods are presented to measure tumor microcirculation by perfusion imaging and elevated interstitial fluid pressure (IFP) using an image-guided robotic system. Measurements are compared to the intra-tumoral accumulation of liposomes, determined using volumetric micro-CT imaging.

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Developmental Biology

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells
Celine L. Bauwens *1, Derek Toms *2, Mark Ungrin 2
1Institute of Biomaterials and Biomedical Engineering, University of Toronto, 2Department of Comparative Biology and Experimental Medicine, University of Calgary

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

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Education

Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by π-π Stacking Interactions
Frantz Le Dévédec 1, Loujin Houdaihed 1, Christine Allen 1
1Leslie Dan Faculty of Pharmacy, University of Toronto

The key steps of living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE) are described. The resulting block copolymer micelles (BCMs) were loaded with doxorubicin 14% (wt%) and sustained release of drug over 4 days under physiologically relevant conditions was obtained.

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Immunology and Infection

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies
Andrzej Chruscinski 1, Flora Y. Y. Huang 1, Antigona Ulndreaj 2, Conan Chua 1, Michael Fehlings 3, Vivek Rao 4, Heather J. Ross 1, Gary A. Levy 1
1Multi-Organ Transplant Program, University Health Network, 2Institute of Medical Science, University of Toronto, 3Divison of Neurosurgery, University Health Network, 4Division of Cardiac Surgery, University Health Network

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

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Immunology and Infection

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses
Jeeyoon Jennifer Ahn *1, Thirumahal Selvanantham *1, Monan Angela Zhang *1, Thierry Mallevaey 1, Shannon E. Dunn 1,2,3
1Department of Immunology, University of Toronto, 2Toronto General Research Institute, University Health Network, 3Women's College Research Institute

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-γ (IFN-γ) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

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Developmental Biology

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System
Enrica Bertin 1, Martina Piccoli 1, Chiara Franzin 1, Andras Nagy 2, Maria Mileikovsky 2, Paolo De Coppi 3, Michela Pozzobon 1
1Stem Cell and Regenerative Medicine Laboratory, Fondazione Istituto di Ricerca Pediatrica Citta della Speranza, 2Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 3Stem Cells and Regenerative Medicine Section, Developmental Biology and Cancer Programme, UCL Institute of Child Health and Great Ormond Street Hospital

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

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Immunology and Infection

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain
Tingxi Guo 1,2, Toshiki Ochi *2, Munehide Nakatsugawa *2, Yuki Kagoya 2, Mark Anczurowski 1,2, Chung-Hsi Wang 1,2, Muhammed A. Rahman 2, Kayoko Saso 2, Marcus O. Butler 1,2, Naoto Hirano 1,2
1Department of Immunology, University of Toronto, 2Princess Margaret Cancer Centre, University Health Network

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCRα or TCRβ of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

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Immunology and Infection

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model
Trevor Beaudoin 1, Sarah Kennedy 2, Yvonne Yau 3, Valerie Waters 4
1Physiology and Experimental Medicine, Hospital for Sick Children, 2Department of Clinical Microbiology, Royal College of Surgeons in Ireland, 3Department of Laboratory Medicine and Pathobiology, University of Toronto, 4Department of Pediatrics, University of Toronto

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

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Developmental Biology

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach
Yun-Ui Bae 1, Hoon-Ki Sung 2,3, Jae-Ryong Kim 1
1Department of Biochemistry and Molecular Biology and Smart-aging Convergence Research Center, College of Medicine, Yeungnam University, 2Physiology and Experimental Medicine Program, The Hospital for Sick Children Research Institute, 3Department of Laboratory Medicine and Pathobiology, University of Toronto

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.

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Immunology and Infection

Use of a Monocyte Monolayer Assay to Evaluate Fcγ Receptor-mediated Phagocytosis
Tik Nga Tong 1, Donald R. Branch 1,2
1Department of Laboratory Medicine and Pathobiology, University of Toronto, 2Centre for Innovation, Canadian Blood Services

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fcγ receptor (FcγR)-mediated phagocytosis.

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Engineering

Solvent Bonding for Fabrication of PMMA and COP Microfluidic Devices
Alwin M. D. Wan 1, Thomas A. Moore 1,2, Edmond W. K. Young 1,2
1Department of Mechanical & Industrial Engineering, University of Toronto, 2Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto

Solvent bonding is a simple and versatile method for fabricating thermoplastic microfluidic devices with high quality bonds. We describe a protocol to achieve strong, optically clear bonds in PMMA and COP microfluidic devices that preserve microfeature details, by a judicious combination of pressure, temperature, an appropriate solvent, and device geometry.

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Medicine

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity (AIDN): A Translational Neuroscience Approach
Emmett E. Whitaker 1,2, Christopher Z. Zheng 1, Bruno Bissonnette 1,2,3, Andrew D. Miller 4, Tanner L. Koppert 1,2, Joseph D. Tobias 1,2, Christopher R. Pierson 5,6, Fedias L. Christofi 1
1Department of Anesthesiology, Ohio State University College of Medicine, 2Department of Anesthesiology and Pain Medicine, Nationwide Children's Hospital, 3Department of Anaesthesia and Critical Care Medicine, University of Toronto, 4Department of Biomedical Sciences, Section of Anatomic Pathology, Cornell University College of Veterinary Medicine, 5Department of Pathology and Anatomy, Ohio State University College of Medicine, 6Department of Pathology and Laboratory Medicine, Nationwide Children's Hospital

Anesthesia-induced developmental neurotoxicity (AIDN) research has focused on rodents, which are not broadly applicable to humans. Non-human primate models are more relevant, but are cost-prohibitive and difficult to use for experimentation. The piglet, in contrast, is a clinically relevant, practical animal model ideal for the study of anesthetic neurotoxicity.

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Developmental Biology

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors
Emily C. McGaugh 1,2,3, M. Cristina Nostro 1,2,3
1Toronto General Research Institute, University Health Network, 2McEwen Centre for Regenerative Medicine, University Health Network, 3Department of Physiology, University of Toronto

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

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Medicine

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents
Martin Sidler 1,2,3, Karen J. Aitken 1, Jia Xin Jiang 4, Darius J. Bägli 1,2,3
1Developmental and Stem Cell Biology, Research Institute, The Hospital for Sick Children, 2Institute of Medical Science, University of Toronto, 3Pediatric Urology, The Hospital for Sick Children, 4Molecular Structure and Function, Research Institute, The Hospital for Sick Children

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

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JoVE Journal

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model
Ann Lee-Barthel 1, Keith Baar 1,2, Daniel W. D. West 1,3
1Department of Neurobiology, Physiology, and Behavior, University of California, Davis, 2Department of Physiology and Membrane Biology, University of California, Davis, 3Faculty of Kinesiology and Physical Education, University of Toronto

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

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Developmental Biology

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation
Xuetao Sun 1, Sara S. Nunes 1,2,3,4
1Toronto General Research Institute, University Health Network, 2Institute of Biomaterials and Biomedical Engineering, University of Toronto, 3Heart & Stroke/Richard Lewar Centre of Excellence, University of Toronto, 4Laboratory of Medicine and Pathobiology, University of Toronto

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

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Developmental Biology

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells
Peter Szaraz *1,2, Yarden S. Gratch *1, Farwah Iqbal 1,2, Clifford L. Librach 1,2,3,4,5
1Create Fertility Centre, 2Department of Physiology, University of Toronto, 3Department of Obstetrics and Gynecology, University of Toronto, 4Department of Physiology, University of Toronto, 5Department of Obstetrics and Gynecology, Women's College Hospital

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

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Developmental Biology

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro
Farwah Iqbal 1,2, Yarden S. Gratch 1, Peter Szaraz 1,2, Clifford L. Librach 1,2,3,4,5
1Create Fertility Centre, 2Department of Physiology, University of Toronto, 3Department of Obstetrics and Gynecology, University of Toronto, 4Department of Medical Sciences, University of Toronto, 5Department of Obstetrics and Gynecology, Women's College Hospital

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

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Biology

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets
Madina Makhmutova 1, Tao Liang 2, Herbert Gaisano 2, Alejandro Caicedo 1, Joana Almaça 1
1Department of Medicine, University of Miami, 2Department of Medicine, University of Toronto

We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

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JoVE Journal

Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid
Shaun Pacheco 1, Shan-Yu Fung 2, Mingyao Liu 1,3
1Latner Thoracic Surgery Research Laboratories, University Health Network, 2Department of Pediatrics, British Columbia Children's Hospital & University of British Columbia, 3Institute of Medical Science, University of Toronto

This protocol describes a clinically-applicable means of dissolving hydrophobic compounds in an aqueous environment using combinations of self-assembling peptide and amino acid solutions. Our method resolves a major limitation of hydrophobic therapeutics, which lack safe, efficient means of solubility and delivery methods into clinical settings.

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Cancer Research

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice
Rui Xue Zhang 1, Tian Zhang 1, King Chen 1, Ji Cheng 1, Paris Lai 1, Andrew M. Rauth 2, K. Sandy Pang 1, Xiao Yu Wu 1
1Department of Pharmaceutical Sciences, University of Toronto, 2Departments of Medical Biophysics and Radiation Oncology, University of Toronto, Ontario Cancer Institute, University Health Network

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

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Biochemistry

Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy
Yulong Sun 1, Philbert Ip 2, Avijit Chakrabartty 1,2
1Department of Medical Biophysics, University of Toronto, 2Department of Biochemistry, University of Toronto

Background autofluorescence of biological samples often complicates fluorescence-based imaging techniques, especially in aged human postmitotic tissues. This protocol describes how autofluorescence from these samples can be effectively removed using a commercially available light emitting diode light source to photobleach the sample prior to immunostaining.

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Genetics

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification
Allison Jandura 1,2, Jack Hu 1, Ronit Wilk 1,2, Henry M. Krause 1,2
1The Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 2Department of Molecular Genetics, University of Toronto

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

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Medicine

MRI-guided Focused Ultrasound Thalamotomy for Patients with Medically-refractory Essential Tremor
Ying Meng 1, Yuexi Huang 2, Benjamin Solomon 2, Kullervo Hynynen 2, Nadia Scantlebury 1, Michael L. Schwartz 1, Nir Lipsman 1
1Division of Neurosurgery, Sunnybrook Health Sciences Centre, 2Sunnybrook Research Institute, Sunnybrook Health Sciences Centre

High-intensity MRI guided focused ultrasound is an emerging noninvasive technique to precisely ablate brain tissue. It has been shown to be safe and effective in treating medically-refractory essential tremor. This article describes the protocol for thalamotomy from patient selection to equipment setup to post-treatment follow-up.

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Developmental Biology

Dissection and Staining of Drosophila Pupal Ovaries
Karen Sophia Park 1, Dorothea Godt 2, Daniel Kalderon 1
1Department of Biological Sciences, Columbia University, 2Department of Cell and Systems Biology, University of Toronto

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

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Environment

Determination of the Settling Rate of Clay/Cyanobacterial Floccules
Tiffany Playter 1, Kurt Konhauser 1, George W. Owttrim 2, Denise S. Whitford 2, Tyler Warchola 1, Cheryl Hodgson 1,3, Aleksandra M. Mloszewska 4, Bruce Sutherland 1, J.-P. Zonneveld 1, S. George Pemberton 1, Murray K. Gingras 1
1Department of Earth and Atmospheric Sciences, University of Alberta, 2Department of Biological Sciences, University of Alberta, 3Department of Earth Sciences, Simon Fraser University, 4Earth Sciences Department, University of Toronto

The interaction and sedimentation of the clay and bacterial cells within the marine realm, observed in natural environments, can be best investigated in a controlled lab environment. Here, we describe a detailed protocol, which outlines a novel method for measuring the sedimentation rate of clay and cyanobacterial floccules.

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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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Immunology and Infection

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity
Ganggang Huang *1, Zhenwei Zhong *1, Shane Miersch 2,3, Sachdev S. Sidhu 1,2,3, Shin-chen Hou 1, Donghui Wu 1
1Laboratory of Antibody Engineering, Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, 2Banting and Best Department of Medical Research, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, 3Department of Molecular Genetics, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto

This protocol describes a detailed procedure for the construction of a phage-displayed synthetic antibody library with tailored diversity. Synthetic antibodies have broad applications from basic research to disease diagnostics and therapeutics.

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Medicine

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae
Tali Farine 1,2, Michael Parsons 2, Stephen Lye 1,2,3, Oksana Shynlova 1,2,3
1Department of Physiology, University of Toronto, 2Lunenfeld-Tanenbaum Research Institute, Sinai Health System, 3Department of Obstetrics and Gynecology, University of Toronto

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

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Neuroscience

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury
Nishanth Lakshman 1,2, Wenjun Xu 2, Cindi M. Morshead 1,2,3
1Institute of Medical Science, University of Toronto, 2Department of Surgery, University of Toronto, 3Institute of Biomaterials and Biomedical Engineering, University of Toronto

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

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JoVE Journal

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques
June Ereño-Orbea *1, Taylor Sicard *1,2, Hong Cui 1, Indira Akula 1, Jean-Philippe Julien 1,2,3
1Program in Molecular Medicine, The Hospital for Sick Children Research Institute, 2Department of Biochemistry, University of Toronto, 3Department of Immunology, University of Toronto

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

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Biology

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge
Oliver A. Kent 1, Charles Steenbergen 2, Samarjit Das 2
1Princess Margaret Cancer Centre, University of Toronto, 2Department of Pathology, Department of Cardiology, Johns Hopkins University

Tissue-specific microRNA inhibition is a technology that is underdeveloped in the microRNA field. Herein, we describe a protocol to successfully inhibit the miR-181 microRNA family in myoblast cells from the heart. Nanovector technology is used to deliver a microRNA sponge that demonstrates significant in vivo cardio-specific miR-181 family inhibition.

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Neuroscience

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue
Michael Chang 1,2, Suzie Dufour 1,3, Peter L. Carlen 1,2,3,5,6, Taufik A. Valiante 1,2,3,4
1Division of Fundamental Neurobiology, Krembil Research Institute, 2Institute of Medical Science, Faculty of Medicine, University of Toronto, 3Institute of Biomaterials and Biomedical Engineering, University of Toronto, 4Division of Neurosurgery, Department of Surgery, University of Toronto, 5Division of Neurology, Department of Medicine, University of Toronto, 6Department of Physiology, University of Toronto

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

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JoVE Core

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex
Pantelis Lioumis 1, Reza Zomorrodi 1, Itay Hadas 1, Zafiris J. Daskalakis 1,2, Daniel M. Blumberger 1,2
1Temerty Centre for Therapeutic Brain Intervention at the Centre for Addiction and Mental Health, 2Department of Psychiatry and Institute of Medical Science, Faculty of Medicine, University of Toronto

The protocol presented here is for TMS-EEG studies utilizing intracortical excitability test-retest design paradigms. The intent of the protocol is to produce reliable and reproducible cortical excitability measures for assessing neurophysiological functioning related to therapeutic interventions in the treatment of neuropsychiatric diseases such as major depression.

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Immunology and Infection

Isolation and Identification of Extravascular Immune Cells of the Heart
Laura Aronoff 1,2, Slava Epelman 1,2,3,4,5, Xavier Clemente-Casares 1,2
1Toronto General Hospital Research Institute, University Health Network (UHN), 2Dept of Laboratory Medicine and Pathobiology, University of Toronto, 3Dept of Immunology, University of Toronto, 4Peter Munk Cardiac Centre, 5Ted Rogers Centre for Heart Research

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

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Medicine

Autonomic Function Following Concussion in Youth Athletes: An Exploration of Heart Rate Variability Using 24-hour Recording Methodology
Melissa Paniccia 1, Tim Taha 2, Michelle Keightley 1,3, Scott Thomas 2, Lee Verweel 1, James Murphy 1, Katherine Wilson 1, Nick Reed 1,3,4
1Concussion Centre, Bloorview Research Institute, Holland Bloorview Kids Rehabilitation Hospital, 2Faculty of Kinesiology and Physical Education, University of Toronto, 3Rehabilitation Sciences Institute, Faculty of Medicine, University of Toronto, 4Department of Occupational Science and Occupational Therapy, Faculty of Medicine, University of Toronto

We demonstrate a 24 h heart rate recording methodology to evaluate the influence of concussion across the recovery trajectory in youth athletes, within an ecologically valid context.

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Bioengineering

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers
H.S. Jeffrey Man 1,2, Aravin N. Sukumar 1,2, Kyung Ha Ku 2,3, Michelle K. Dubinsky 1,2, Noeline Subramaniam 1,2, Philip A. Marsden 1,2,3,4
1Institute of Medical Science, University of Toronto, 2Keenan Research Centre in the Li Ka Shing Knowledge Institute, St. Michael's Hospital, 3Department of Laboratory Medicine and Pathobiology, University of Toronto, 4Department of Medicine, University of Toronto

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

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Biology

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining
Yanqing Jiang *1, Joanna Lan-Hing Yeung *1, Ju Hee Lee 1,2, James An 1, Patrick E. Steadman 3, Jae-Ryong Kim 4, Hoon-Ki Sung 1,2,5
1Translational Medicine Program, The Hospital for Sick Children, 2Department of Laboratory Medicine and Pathobiology, University of Toronto, 3Neurosciences & Mental Health Program, The Hospital for Sick Children, 4Department of Biochemistry and Molecular Biology, Smart-Aging Convergence Research Center, College of Medicine, Yeungnam University, 5Banting and Best Diabetes Centre, University of Toronto

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

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Bioengineering

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles
Hisato Konoeda 1, Hong Yang 2, Chengliang Yang 1, Annette Gower 1, Chun Xu 1, Wei Zhang 1, Mingyao Liu 1,3
1Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, 2Respiratory Medicine Research Laboratory, Institute of Translation Medicine, Shanghai General Hospital, Shanghai Jiaotong University, 3Institute of Medical Science, Faculty of Medicine, University of Toronto

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

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Biochemistry

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation
Ying Fan *1, Francesca Tonelli *1, Shalini Padmanabhan 2, Marco A.S. Baptista 2, Lindsey Riley 2, Danielle Smith 3, Connie Marras 4, Andrew Howden 5, Dario R. Alessi 1, Esther Sammler 1,6
1MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, 2The Michael J. Fox Foundation for Parkinson's Research, 3Department of Medical and Molecular Genetics, Indiana University School of Medicine, 4Morton and Gloria Shulman Movement Disorders Centre and the Edmond J. Safra Program in Parkinson's Disease, Toronto Western Hospital, University of Toronto, 5Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, 6Division of Molecular and Clinical Medicine, School of Medicine, Ninewells Hospital and Medical School, University of Dundee

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson’s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

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Medicine

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation
Roberto V. P. Ribeiro 1,2, Juglans S. Alvarez 1, Frank Yu 1, Mitchell B. Adamson 1,2, Naoto Fukunaga 1, Cyril Serrick 3, Ved Bissoondath 1, Massimiliano Meineri 4,5, Mitesh V. Badiwala 1,6, Vivek Rao 1,2,6
1Division of Cardiovascular Surgery, Peter Munk Cardiac Center, Toronto General Hospital, University Health Network, 2Institute of Medical Science, University of Toronto, 3Perfusion and Anesthesia Services, Toronto General Hospital, University Health Network, 4Department of Anesthesia and Pain Management, Toronto General Hospital, University Health Network, 5Department of Anesthesia, University of Toronto, 6Department of Surgery, Faculty of Medicine, University of Toronto

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

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Cancer Research

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells
Lauren Philp 1,2, Harley Chan 3, Marjan Rouzbahman 4, Ariana Rostami 5, Lili Ding 6, Scott V. Bratman 5,6, Margarete K. Akens 5,7,8, Jonathan C. Irish 7,9,11, Marcus Q. Bernardini 10, Gang Zheng 5,11
1Institute of Medical Science, University of Toronto, 2Department of Obstetrics and Gynecology, University of Toronto, 3Guided Therapeutics Laboratory, TECHNA Institute, University Health Network, 4Department of Pathology, Princess Margaret Cancer Center, University Health Network, 5Department of Medical Biophysics, University of Toronto, 6Princess Margaret Cancer Center, University Health Network, 7Techna Institute, University Health Network, 8Department of Surgery, University of Toronto, 9Department of Otolaryngology, Head and Neck Surgery, University of Toronto, 10Division of Gynecologic Oncology, University of Toronto /Princess Margaret Cancer Center, University Health Network, 11Princess Margaret Cancer Center, University Health Network

This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

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Immunology and Infection

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils
Jeffrey W. Chadwick 1,2, Michael Glogauer 1,2
1Department of Dental Oncology and Maxillofacial Prosthetics, Princess Margaret Cancer Centre, University Health Network, 2Faculty of Dentistry, University of Toronto

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

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Medicine

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis
Kaiyue Zhang 1, Chen Wang 1, Ran Wang 2, Shang Chen 1, Zongjin Li 1
1Nankai University School of Medicine, 2State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

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Genetics

Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Katherine Chan *1, Amy Hin Yan Tong *1, Kevin R Brown 1, Patricia Mero 1, Jason Moffat 1,2,3
1Donnelly Centre, University of Toronto, 2Department of Molecular Genetics, University of Toronto, 3Institute for Biomaterials and Biomedical Engineering, University of Toronto

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

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Medicine

Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch
Yaguang Li 1, Junhong Liu 2,3, Gaixia Liu 1, Zhenhong Pan 2,3, Mingxia Zhang 1, Yao Ma 4, Qingxia Wei 1,5, Hongping Xia 1,6, Rui Xue Zhang 1,2, Junjun She 1
1Department of General Surgery, The First Affiliated Hospital, Xi'an Jiaotong University, 2Institute of Medical Research, Northwestern Polytechnical University, 3School of Life Sciences, Northwestern Polytechnical University, 4Department of Medicine, Xi'an Jiaotong University, 5Princess Margaret Cancer Center, University of Health Network, 6Department of Pathology, Nanjing Medical University

The presented protocol describes a facile surgical removal of the appendix (caecal patch) in a mouse followed by the induction of inflammatory bowel disease-associated colorectal cancer. This murine appendectomy model enables investigation of the biological role of the appendix in the pathogenesis of human gastrointestinal disease.

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Biology

Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice
Ri Youn Kim *1,2, Ju Hee Lee *3,4, Yena Oh 1,2, Hoon-Ki Sung 3,4,5, Kyoung-Han Kim 1,2
1University of Ottawa Heart Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Translational Medicine Program, The Hospital for Sick Children, 4Department of Laboratory Medicine and Pathobiology, University of Toronto, 5Banting and Best Diabetes Centre, University of Toronto

The current article describes a detailed protocol for isocaloric 2:1 intermittent fasting to protect and treat against obesity and impaired glucose metabolism in wild-type and ob/ob mice.

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Chemistry

Resolving Water, Proteins, and Lipids from In Vivo Confocal Raman Spectra of Stratum Corneum through a Chemometric Approach
Lesheng Zhang *1, Tom Cambron *2, Yueqing Niu 1, Zigang Xu 3, Ning Su 4, Hongyan Zheng 4, Karl Wei 2, Paula Ray 2
1Procter and Gamble, Beijing Innovative center, 2Procter and Gamble, Mason Business Center, 3Department of Dermatology, Beijing Children's Hospital, 4Chinese Academy of Inspection and Quarantine

Here, we present a protocol for collection of confocal Raman spectra from human subjects in clinical studies combined with chemometric approaches for spectral outlier removal and the subsequent extraction of key features.

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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

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation
Xu-Chun Chen *1, Manmeet Sekhon *1,2, Xue-Zhong Ma *1, Justin Manuel 1, Sai Chung 1,2, Eddie He 1, Agata Bartczak 1, Sandra Fischer 3, Cornelia Thoeni 3, Graziano Oldani 1, Catia T. Perciani 1, Sonya MacParland 1,2,3, Ian McGilvray 1
1Multi-Organ Transplant Program, University Health Network, 2Department of Immunology, University of Toronto, 3Department of Laboratory Medicine and Pathobiology, University of Toronto

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver's common hepatic artery to the recipient liver's proper hepatic artery.

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Genetics

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice
Hirofumi Nishizono *1,2,3, Mohamed Darwish *4,5, Hideki Uosaki 6,7, Nanami Masuyama 8,9,10, Motoaki Seki 8,11, Hiroyuki Abe 3, Nozomu Yachie 8,9,10,12,13, Ryohei Yasuda 1
1Max Planck Florida Institute for Neuroscience, 2Life Science Research Center, University of Toyama, 3Department of Biochemical Engineering, Graduate School of Science and Engineering, Yamagata University, 4Graduate School of Innovative Life Science, University of Toyama, 5Department of Biochemistry, Faculty of Pharmacy, Cairo University, 6Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, 7Division of Stem Cell Research and Drug Development, Center for Development of Advanced Medical Technology, Jichi Medical University, 8Synthetic Biology Division, Research Center for Advanced Science and Technology, University of Tokyo, 9Institute for Advanced Biosciences, Keio University, 10Graduate School of Media and Governance, Keio University, 11Department of Molecular Oncology, Graduate School of Medicine, Chiba University, 12Department of Biological Sciences, School of Science, University of Tokyo, 13College of Arts and Sciences, University of Tokyo

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

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Medicine

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes
Yun Liu *1, Yin Liang *1, Mengxue Wang *1, Chen Wang 1, Heng Wei 2, Keiji Naruse 1, Ken Takahashi 1
1Department of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

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Genetics

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)
Kaitlin N. Montanera 1,2, Ho Sung Rhee 1,2
1Department of Cell and Systems Biology, University of Toronto, 2Department of Biology, University of Toronto

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

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Biology

Quantification of Circulating Pig-Specific DNA in the Blood of a Xenotransplantation Model
Yangyang Deng *1,2, Ming Zhou *1,3, Ying Lu 1, Jiao Chen 1, Zuhui Pu 4, Dongjing Yu 1,5, Yifan Dai 6, Yongqiang Zhan 7, Lisha Mou 1
1Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen University Health Science Center, Shenzhen University School of Medicine, First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, 2Xianning Hospital of Traditional Chinese Medicine, 3Liver-biotechnology (Shenzhen) Co., Ltd., 4Department of Radiology, Shenzhen University Health Science Center, Shenzhen University School of Medicine, First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, 5Department of Life Sciences, University of Toronto, 6Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, 7Department of Hepatopancreatobiliary Surgery, Shenzhen University Health Science Center, Shenzhen University School of Medicine, First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital

In this protocol, porcine specific primers were designed, plasmids-containing porcine specific DNA fragments were constructed, and standard curves for quantitation were established. Using species-specific primers, cpsDNA was quantified by qPCR in pig-to-mouse cell transplantation models and pig-to-monkey artery patch transplantation models.

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Genetics

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity
Sampath Kumar Loganathan 1, Ahmad Malik 1,2, Ellen Langille 1,2, Chen Luxenburg 3, Daniel Schramek 1,2
1Centre for Molecular and Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 2Department of Molecular Genetics, University of Toronto, 3Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University

Here we describe a rapid and direct in vivo CRISPR/Cas9 screening methodology using ultrasound-guided in utero embryonic lentiviral injections to simultaneously assess functions of several genes in the skin and oral cavity of immunocompetent mice.

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Biology

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat
Lucas Jaryd Iringan Te *1, Christina Doherty *1, Judy Correa 1, Jane Batt 1,2
1Keenan Research Center for Biomedical Science, St Michaels Hospital, Unity Health Toronto, 2Department of Medicine and Interdepartmental Division of Critical Care Medicine, University of Toronto

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

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Education

Blood Flow Imaging with Ultrafast Doppler
Jerome Baranger 1,2, Luc Mertens 1,2, Olivier Villemain 1,2,3
1Translational Medicine Department, The Hospital for Sick Children, PGCRL Research Institute, 2The Labatt Family Heart Centre, Department of Pediatric, The Hospital for Sick Children, University of Toronto, 3Medical Biophysics Department, University of Toronto

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every ≈0.3 ms (depending on the ultrasound time of flight).

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Bioengineering

Capturing Representative Hand Use at Home Using Egocentric Video in Individuals with Upper Limb Impairment
Meng-Fen Tsai 1,3, Andrea Bandini 3, Rosalie H. Wang 2,3,5, José Zariffa 1,3,4,5
1Institute of Biomedical Engineering, University of Toronto, 2Department of Occupational Science and Occupational Therapy, University of Toronto, 3KITE, Toronto Rehabilitation Institute, University Health Network, 4Edward S. Rogers Sr. Department of Electrical and Computer Engineering, University of Toronto, 5Rehabilitation Sciences Institute, University of Toronto

A protocol is proposed to capture natural hand function of individuals with hand impairments during their daily routines using an egocentric camera. The goal of the protocol is to ensure that the recordings are representative of an individual's typical hand use during activities of daily living at home.

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Medicine

Human Fetal Blood Flow Quantification with Magnetic Resonance Imaging and Motion Compensation
Datta Singh Goolaub 1,2, Davide Marini 3,4, Mike Seed 4,5, Christopher K. Macgowan 1,2
1Department of Medical Biophysics, University of Toronto, 2Division of Translational Medicine, The Hospital for Sick Children, 3Labatt Family Heart Centre, The Hospital for Sick Children, 4Department of Pediatrics, University of Toronto, 5Division of Pediatric Cardiology, The Hospital for Sick Children

Here we present a protocol for measuring fetal blood flow rapidly with MRI and retrospectively performing motion correction and cardiac gating.

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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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Bioengineering

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation
Ryan C. Lee 1, Travis R. Douglas 1, Leo Y. T. Chou 1
1Institute of Biomedical Engineering, University of Toronto

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

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Medicine

A Large Animal Model for Acute Kidney Injury by Temporary Bilateral Renal Artery Occlusion
Ilias P. Doulamis 1, Alvise Guariento 1, Mossab Y. Saeed 1, Rio S. Nomoto 1, Thomas Duignan 1, Pedro J. del Nido 1, James D. McCully 1
1Department of Cardiac Surgery, Boston Children’s Hospital, Harvard Medical School

This study presents a highly reproducible large animal model of renal ischemia-reperfusion injury in swine using temporary percutaneous bilateral balloon-catheter occlusion of the renal arteries for 60 min and reperfusion for 24 h.

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Bioengineering

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability
Charissa Poon 1,2, Melina Mühlenpfordt *3, Marieke Olsman *3, Spiros Kotopoulis 4,5, Catharina de Lange Davies 3, Kullervo Hynynen 1,2,6
1Physical Sciences Platform, Sunnybrook Research Institute, 2Institute of Biomedical Engineering, University of Toronto, 3Department of Physics, Norwegian University of Science and Technology, 4Department of Clinical Medicine, University of Bergen, 5Exact Therapeutics AS, 6Department of Medical Biophysics, University of Toronto

This protocol describes the surgical and technical procedures that enable real-time in vivo multiphoton fluorescence imaging of the rodent brain during focused ultrasound and microbubble treatments to increase blood-brain barrier permeability.

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Bioengineering

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues
Heta Lad 1,2, Brennen Musgrave 1,2, Majid Ebrahimi 1,2, Penney M. Gilbert 1,2,3
1Institute of Biomedical Engineering, University of Toronto, 2Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 3Department of Cell and Systems Biology, University of Toronto

This manuscript describes a detailed protocol to produce arrays of 3D human skeletal muscle microtissues and minimally invasive downstream in situ assays of function, including contractile force and calcium handling analyses.

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Neuroscience

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations
Samantha Ing-Esteves 1,2, Julie L. Lefebvre 1,2
1Program for Neuroscience and Mental Health, Hospital for Sick Children, 2Department of Molecular Genetics, University of Toronto

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

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Biology

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading
Ernest Iu *1, Alexander Bogatch *1, Sergey V. Plotnikov 1
1Department of Cell and Systems Biology, University of Toronto

In this protocol, we present the experimental procedures of a cell spreading assay that is based on live-cell microscopy. We provide an open-source computational tool for the unbiased segmentation of fluorescently labeled cells and quantitative analysis of lamellipodia dynamics during cell spreading.

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Biology

Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells
Magdalena M. Szewczyk 1, Victoria Vu 1, Dalia Barsyte-Lovejoy 1,2
1Structural Genomics Consortium, University of Toronto, 2Department of Pharmacology and Toxicology, University of Toronto

These protocols provide the methodology used to assess the enzymatic activity of individual members of the protein arginine methyltransferase (PRMT) family in cells. Detailed guidelines on assessing PRMT activity using endogenous and exogenous biomarkers, methyl-arginine recognizing antibodies, and inhibitor tool compounds are described.

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Bioengineering

Control of Cell Geometry through Infrared Laser Assisted Micropatterning
Shuying Yang 1, Chen Tuo 1, Ernest Iu 1, Sergey V. Plotnikov 1
1Department of Cell and Systems Biology, University of Toronto

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

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Biochemistry

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System
Ashley Hutchinson 1, Almagul Seitova 1
1Structural Genomics Consortium, University of Toronto

The baculovirus expression vector system (BEVS) is a robust platform for expression screening and production of protein arginine methyltransferases (PRMTs) to be used for biochemical, biophysical, and structural studies. Milligram quantities of material can be produced for the majority of PRMTs and other proteins of interest requiring a eukaryotic expression platform.

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Medicine

The Use of 3D Echocardiography in Surgical Planning of the Mitral Valve in Pediatric Cardiology
Nick Arbic 1, Andreea Dragulescu 1, Luc Mertens 1, Olivier Villemain 1
1Division of Cardiology, Department of Pediatrics, The Hospital for Sick Children, University of Toronto

3D echocardiography of the mitral valve in pediatric cardiology produces full anatomic reconstructions that contribute to improved surgical management. Here, we outline a protocol for 3D acquisition and post-processing of the mitral valve in pediatric cardiology.

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Behavior

Handling Techniques to Reduce Stress in Mice
Michael Marcotte 1, Ashley Bernardo 1,2, Nathaniel Linga 3, Carmina A. Pérez-Romero 4, Jean-Louis Guillou 5, Etienne Sibille 1,2,3, Thomas D. Prevot 1,2
1Campbell Family Mental Health Research Institute of CAMH, 2Department of Psychiatry, University of Toronto, 3Department of Pharmacology and Toxicology, University of Toronto, 4Departamento de Investigación, Universidad Central de Queretaro, 5Centre National de la Recherche Scientifique, UMR 5287, Institut de Neurosciences Cognitives et Intégratives d'Aquitaine, Université de Bordeaux

This paper describes a handling technique in mice, the 3D-handling technique, which facilitates routine handling by reducing anxiety-like behaviors and presents details on two existing related techniques (tunnel and tail handling).

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Medicine

Synthesis and Characterization of Multi-Modal Phase-Change Porphyrin Droplets
Kimoon Yoo 1,2, Alexander Dhaliwal 1,2, Juan Chen 1, Paul S. Sheeran 3, Gang Zheng 1,2
1Princess Margaret Cancer Centre, 2Department of Medical Biophysics, University of Toronto, 3Philips Healthcare

In this protocol, methods for synthesizing and characterizing multi-modal phase-change porphyrin droplets are outlined.

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Immunology and Infection

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression
Kevin Guttman 1, Pauline Wang 2, Lindsay Jackson 1, Amanda Morris 1, Yvonne Yau 3, Valerie Waters 1,4
1Translational Medicine, Hospital for Sick Children, 2Department of Cell and Systems Biology, University of Toronto, 3Microbiology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, 4Infectious Diseases, Department of Pediatrics, Hospital for Sick Children

This protocol presents a method to isolate RNA from Pseudomonas aeruginosa biofilms grown in chamber slides for high throughput sequencing.

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Medicine

Microsurgical Skills of Establishing Permanent Jugular Vein Cannulation in Rats for Serial Blood Sampling of Orally Administered Drug
Weijia Lu *1, Ruimin Miao *1, Sijun Hu 1, Junhong Liu 1, Fanqi Jin 1, Rui Xue Zhang 1
1Xi'an Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Medical Research, Northwestern Polytechnical University

Detailed microsurgical techniques are demonstrated to establish a longer-term jugular vein cannulation rat model for sequential blood collection in the same animal. Physiological and hematological parameters have been monitored during the rat's recovery phase. This model has been applied to study pharmacokinetics of orally administered polyphenol without inducing animal stress.

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Medicine

A Rodent Model of The Ross Operation: Syngeneic Pulmonary Artery Graft Implantation in A Systemic Position
Arben Dedja *1, Claudia Cattapan *1, Giovanni Di Salvo 2, Martina Avesani 2, Jolanda Sabatino 2, Alvise Guariento 1,3, Vladimiro Vida 1
1Pediatric and Congenital Cardiac Surgery Unit, Department of Cardiac, Thoracic and Vascular Sciences and Public Health, University of Padua, 2Pediatric Cardiology Unit, Departments of Women’s and Children’s Health, University of Padua, 3Labatt Family Heart Centre, Department of Cardiovascular Surgery, The Hospital for Sick Children, University of Toronto

We demonstrate how to establish a murine model of pulmonary root implantation into the descending aorta to simulate the Ross procedure. This model enables the medium/long-term evaluation of pulmonary autograft remodeling in a systemic position, representing the basis of developing therapeutic strategies to promote its adaptation.

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Bioengineering

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics
Katariina Jaenes *1, Severino Jefferson Ribeiro da Silva *1,2, Justin R. J. Vigar *1, Kaiyue Wu 3,4, Masoud Norouzi 1, Pouriya Bayat 1, Margot Karlikow 1, Seray Cicek 1, Yuxiu Guo 1, Alexander A. Green 3,4, Lindomar Pena 2, Keith Pardee 1,5
1Leslie Dan Faculty of Pharmacy, University of Toronto, 2Laboratory of Virology and Experimental Therapy (LAVITE), Department of Virology, Aggeu Magalhães Institute (IAM), Oswaldo Cruz Foundation (Fiocruz), 3Department of Biomedical Engineering, Boston University, 4Molecular Biology, Cell Biology & Biochemistry Program, Graduate School of Arts and Sciences, Boston University, 5Department of Mechanical and Industrial Engineering, University of Toronto

Access to decentralized, low-cost, and high-capacity diagnostics that can be deployed into the community for decentralized testing is critical for combating global health crises. This manuscript describes how to build paper-based diagnostics for viral RNA sequences that can be detected with a portable optical reader.

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JoVE Core

Conducting Respiratory Oscillometry in an Outpatient Setting
Ehren Chang 1, Anastasiia Vasileva 1, Cynthia Nohra 1, Clodagh M. Ryan 1, Chung-Wai Chow 1, Joyce Ka Yan Wu 1
1Division of Respirology, Department of Medicine, University Health Network, University of Toronto

We demonstrate a standard operating protocol to conduct respiratory oscillometry, highlighting key quality control and assurance procedures.

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Immunology and Infection

Culturing Lymphocytes in Simulated Microgravity Using a Rotary Cell Culture System
Marieke de Korte 1,2, Armand Keating 1,2,4, Chen Wang 1,3
1Laboratory Medicine and Pathobiology, Temerty Faculty of Medicine, University of Toronto, 2Krembil Research Institute, Toronto Western Hospital, University Health Network, 3Pathology and Lab Medicine, Hematopathology, Mount Sinai Hospital, Sinai Health Systems, 4Medical Oncology and Hematology, Princess Margaret Cancer Centre

This is a step-by-step guide for using a commercially available rotary cell culture system to culture lymphocytes in simulated microgravity using specialized disposable culture vessels. This culturing method may be applied to any suspension-type cell culture.

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Biology

Culturing and Screening the Plant Parasitic Nematode Ditylenchus dipsaci
Savina R. Cammalleri 1,2,3, Jessica Knox 1,3, Peter J. Roy 1,3,4
1Department of Molecular Genetics, University of Toronto, 2Department of Biochemistry, University of Toronto, 3The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 4Department of Pharmacology and Toxicology, University of Toronto

The present protocol describes a reliable and straightforward method for culturing, collecting, and screening Ditylenchus dipsaci.

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Biology

A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines
Sunny Xia 1,3, Michelle Di Paola 3, Nicola L. Jones 2,4,5, Christine E. Bear 1,3,5
1Molecular Medicine, Hospital for Sick Children, 2Cell Biology, Hospital for Sick Children, 3Department of Physiology, University of Toronto, 4Department of Paediatrics, University of Toronto, 5Department of Biochemistry, University of Toronto

This protocol describes a method for the study of electrogenic membrane proteins by measuring changes in membrane potential. This assay provides a platform for the functional readout of multiple ion channels endogenously expressed in epithelial cell lines.

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Immunology and Infection

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection
Alexandra R. Willis 1, Hala Tamim El Jarkass 1, Aaron W. Reinke 1
1Department of Molecular Genetics, University of Toronto

The infection of Caenorhabditis elegans by the microsporidian parasite Nematocida parisii enables the worms to produce offspring that are highly resistant to the same pathogen. This is an example of inherited immunity, a poorly understood epigenetic phenomenon. The present protocol describes the study of inherited immunity in a genetically tractable worm model.

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Medicine

Large-Animal Model of Donation after Circulatory Death and Normothermic Regional Perfusion for Cardiac Assessment
Khalil Khalil *1,2, Roberto V. P. Ribeiro *3, Julgans S. Alvarez 4,5, Mitesh V. Badiwala 4,5, Shant Der Sarkissian 1,2, Nicolas Noiseux 1,2
1Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), 2Department of Surgery, Faculty of Medicine, Université de Montréal, 3Division of Cardiac Surgery, Dalhousie University, 4Division of Cardiovascular Surgery, Peter Munk Cardiac Centre, Toronto General Hospital, 5Department of Surgery, University of Toronto

The protocol describes a large-animal (porcine) model of donation after circulatory death, followed by thoracoabdominal normothermic regional perfusion that closely simulates the clinical scenario in heart transplantation, and has the potential to facilitate therapeutic studies and strategies.

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Bioengineering

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor
Michael S. Xu 1, Golnaz Karoubi 1,2, Thomas K. Waddell 1,3, Siba Haykal 1,4
1Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, 2Institute of Laboratory Medicine and Pathobiology, University of Toronto, 3Division of Thoracic Surgery, Department of Surgery, University of Toronto, 4Division of Plastic and Reconstructive Surgery, Department of Surgery, University of Toronto

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

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Bioengineering

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation
Aisha Adil 1,2, Golnaz Karoubi 1,3, Siba Haykal 1,2,4
1Latner Thoracic Research Surgical Laboratories, University Health Network, Toronto General Hospital, 2Institute of Medical Science, Temerty Faculty of Medicine, University of Toronto, 3Institute of Laboratory Medicine and Pathobiology, Temerty Faculty of Medicine, University of Toronto, 4Division of Plastic & Reconstructive Surgery, Department of Surgery, University of Toronto

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

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Cancer Research

Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model
Claire Wunker 1,2, Karolina Piorkowska 3, Ben Keunen 3, Yael Babichev 2, Suzanne M. Wong 3,4, Maximilian Regenold 5, Michael Dunne 5, Julia Nomikos 1,2, Maryam Siddiqui 6, Samuel Pichardo 6, Warren Foltz 7, Adam C. Waspe 3,8, Justin T. Gerstle 3,9, James M. Drake 1,3,4,10, Rebecca A. Gladdy 1,2,10
1Institute of Medical Science, University of Toronto, 2Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 3The Wilfred and Joyce Posluns Centre for Image-Guided Innovation and Therapeutic Intervention, The Hospital for Sick Children, 4Institute of Biomedical Engineering, University of Toronto, 5Leslie Dan Faculty of Pharmacy, University of Toronto, 6Departments of Radiology and Clinical Neurosciences, University of Calgary, 7Department of Radiation Oncology, University of Toronto, 8Department of Medical Imaging, University of Toronto, 9Department of Pediatric Surgery, University of Toronto, 10Department of Surgery, University of Toronto

Presented here is a protocol to use controlled hyperthermia, generated by magnetic resonance-guided high intensity focused ultrasound, to trigger drug release from temperature-sensitive liposomes in a rhabdomyosarcoma mouse model.

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Studying Protein Arginine Methylation: Approaches and Methods
Dalia Barsyte-Lovejoy 1
1Pharmacology and Toxicology and Structural Genomics Consortium, University of Toronto

Studying Protein Arginine Methylation: Approaches and Methods

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Advanced Techniques for Studying Blood Flow
Jill J. Weyers 1,2, Nilesh R. Ghugre 1,3,4
1Physical Sciences Platform, Sunnybrook Research Institute, 2Institute of Biomedical Engineering, University of Toronto, 3Schulich Heart Research Program, Sunnybrook Research Institute, 4Department of Medical Biophysics, University of Toronto

Advanced Techniques for Studying Blood Flow

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Neuroscience

BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the Rodent Brain
Akiko Sumitomo 1, Runhao Zhou 1,2, Thomas Prevot 1,3, Etienne Sibille 1,2,3, Toshifumi Tomoda 1,3
1Centre for Addiction and Mental Health (CAMH), Campbell Family Mental Health Research Institute, 2Department of Pharmacology and Toxicology, University of Toronto, 3Department of Psychiatry, University of Toronto

The BS3 chemical crosslinking assay reveals reduced cell surface GABAA receptor expression in mouse brains under chronic psychosocial stress conditions.

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Neuroscience

Quantification of Visual Feature Selectivity of the Optokinetic Reflex in Mice
Jiashu Liu 1,2, Bao-hua Liu 1,2
1Department of Biology, University of Toronto Mississauga, 2Department of Cell and Systems Biology, University of Toronto

Here, we describe a standard protocol for quantifying the optokinetic reflex. It combines virtual drum stimulation and video-oculography, and thus allows precise evaluation of the feature selectivity of the behavior and its adaptive plasticity.

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Genetics

Analysis of Transgenerational Epigenetic Inheritance in C. elegans Using a Fluorescent Reporter and Chromatin Immunoprecipitation (ChIP)
Chengyin Li *1, Phoebe A. W. Bhagoutie *1, Victor Lao 1, Arneet L. Saltzman 1
1Department of Cell and Systems Biology, University of Toronto

This protocol describes an RNA interference and ChIP assay to study the epigenetic inheritance of RNAi-induced silencing and associated chromatin modifications in C. elegans.

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Medicine

Measuring Diaphragm Thickness and Function Using Point-of-Care Ultrasound
Catherine A. Bellissimo 1, Idunn S. Morris 2,3,4, Jenna Wong 1, Ewan C. Goligher 1,2,3,5
1Toronto General Hospital Research Institute, 2Interdepartmental Division of Critical Care Medicine, University of Toronto, 3Department of Physiology, Faculty of Medicine, University of Toronto, 4Deparatment of Intensive Care Medicine, Nepean Hospital, 5Division of Respirology, Department of Medicine, University Health Network

Diaphragm thickness and function can be assessed in healthy individuals and critically ill patients using point-of-care ultrasound. This technique offers an accurate, reproducible, feasible, and well-tolerated method for evaluating diaphragm structure and function.

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Immunology and Infection

Scoring Central Nervous System Inflammation, Demyelination, and Axon Injury in Experimental Autoimmune Encephalomyelitis
Carmen C. Ucciferri 1, Annette Gower 2, Nuria Alvarez-Sanchez 1,2, Heather Whetstone 3, Valeria Ramaglia 1, Jennifer L. Gommerman 1, Koroboshka Brand-Arzamendi 2, Raphael Schneider 2,4, Shannon E Dunn 1,2,5
1Department of Immunology, University of Toronto, 2Keenan Research Centre for Biomedical Science of St. Michael’s Hospital, 3Sickkids Research Institute, The Hospital for Sick Children, 4BARLO MS Centre, St. Michael’s Hospital, 5Women’s College Research Institute, Women’s College Hospital

Experimental autoimmune encephalomyelitis (EAE) serves as an animal model of multiple sclerosis. This article describes an approach for scoring spinal cord inflammation, demyelination, and axonal injury in EAE. Additionally, a method to quantify soluble neurofilament light levels in the mice serum is presented, facilitating the assessment of axonal injury in live mice.

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Medicine

Murine Intrapulmonary Tracheal Transplantation: A Model for Investigating Obliterative Airway Disease After Lung Transplantation
Yamato Suzuki 1,2, Stephen Juvet 1,2, Mingyao Liu 1,2, Shaf Keshavjee 1,2
1Latner Thoracic Research Laboratories, Toronto General Hospital Research Institute, University Health Network, 2Temerty Faculty of Medicine, University of Toronto

The murine intrapulmonary tracheal transplantation (IPTT) model is valuable for studying obliterative airway disease (OAD) after lung transplantation. It offers insights into lung-specific immunological and angiogenic behavior in airway obliteration after allotransplantation with high reproducibility. Here, we describe the IPTT procedure and its expected results.

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Medicine

Establishment of an Ex Vivo Lung Perfusion Rat Model for Translational Insights in Lung Transplantation
Paolo Oliveira *1,3, Keiji Yamanashi *1, Aizhou Wang 1, Marcelo Cypel 1,2
1Toronto General Hospital Research Institute, University Health Network, 2Department of Surgery, University of Toronto, 3Departamento de Cardiopneumologia, Instituto do Coração, Faculdade de Medicina HCFMUSP, Universidade de Sao Paulo

Here, we describe the necessary steps for establishing a rat EVLP model and show the inflammatory profile associated with the perfused lungs. The aim is to propagate knowledge and experiences about the rat EVLP model, enabling the integral understanding of the biological responses associated with that revolutionary technique.

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Cancer Research

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research
W. Jeffrey Zabel *1, Nader Allam *1, Hector Alejandro Contreras Sanchez 1, Warren Foltz 2,3, Costel Flueraru 4, Edward Taylor 2,3, Alex Vitkin 1,2,3
1Department of Medical Biophysics, University of Toronto, 2Radiation Medicine Program, Princess Margaret Cancer Centre, 3Department of Radiation Oncology, University of Toronto, 4Advanced Electronic and Photonics Research Center, National Research Council of Canada

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

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Neuroscience

A Visual Approach for Inducing Dolichoectasia in Mice to Model Large Vessel-Mediated Cerebrovascular Dysfunction
Dominic Simpson 1,2, Christopher D. Morrone 1, Darcy Wear 1,2, Jose Gutierrez 3, Wai Haung Yu 2
1Brain Health Imaging Centre, Centre for Addiction and Mental Health, 2Department of Pharmacology and Toxicology, University of Toronto, 3Department of Neurology, Columbia University

We demonstrate chemically inducing large blood vessel dilatation in mice as a model for investigating cerebrovascular dysfunction, which can be used for vascular dementia and Alzheimer's disease modeling. We also demonstrate visualizing the vasculature by injecting silicone rubber compound and providing clear visual guidance for measuring changes in blood vessel size.

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