University of Toronto

36 articles published in JoVE

Template Directed Synthesis of Plasmonic Gold Nanotubes with Tunable IR Absorbance

JoVE 50420 4/01/2013

Colin R. Bridges, Tyler B. Schon, Paul M. DiCarmine, Dwight S. Seferos

Department 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.

Bringing the Visible Universe into Focus with Robo-AO

JoVE 50021 2/12/2013

Christoph Baranec^1,2, Reed Riddle¹, Nicholas M. Law³, A.N. Ramaprakash⁴, Shriharsh P. Tendulkar², Khanh Bui¹, Mahesh P. Burse⁴, Pravin Chordia⁴, Hillol K. Das⁴, Jack T.C. Davis¹, Richard G. Dekany¹, Mansi M. Kasliwal⁵, Shrinivas R. Kulkarni^1,2, Timothy D. Morton², Eran O. Ofek⁶, Sujit Punnadi⁴

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.

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

JoVE 50066 12/17/2012

Xianying A. Cui, Alexander F. Palazzo

Department 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.

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

JoVE 4072 12/15/2012

Yo Suzuki¹, Jason Stam¹, Mark Novotny², Nozomu Yachie³, Roger S. Lasken², 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.

C. elegans Tracking and Behavioral Measurement

JoVE 4094 11/17/2012

Jirapat Likitlersuang¹, Greg Stephens^2,3, Konstantine Palanski¹, 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.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

JoVE 4057 11/12/2012

Mohan Babu^1,2, Olga Kagan¹, Hongbo Guo¹, 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.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

JoVE 4056 11/12/2012

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.

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

JoVE 4193 10/13/2012

Robart Babona-Pilipos¹, Milos R. Popovic², Cindi M. Morshead³

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.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

JoVE 4417 9/20/2012

Richard W. Loo^1,2, Jane Betty Goh^1,2, Calvin C.H. Cheng¹, Ning Su¹, 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.

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

JoVE 4041 7/31/2012

Halil Aydin*, Farshad C. Azimi*, Jonathan D. Cook*, Jeffrey E. Lee

Department 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.

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

JoVE 4014 7/23/2012

Rahul Kushwah¹, Jim Hu²

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.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

JoVE 3874 5/16/2012

Aida Gordanpour¹, Robert K. Nam², Linda Sugar³, Stephanie Bacopulos¹, 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.

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

JoVE 3932 3/31/2012

Faiza Waheed^1,2, Pamela Speight^1,2, Qinghong Dan^1,2, Rafael Garcia-Mata³, 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.

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

JoVE 3555 3/13/2012

Meaghan A. O'Reilly¹, 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 brain^1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Competitive Genomic Screens of Barcoded Yeast Libraries

JoVE 2864 8/11/2011

Andrew M. Smith*^1,2, Tanja Durbic*^2,3, Julia Oh*⁴, Malene Urbanus^1,2, Michael Proctor⁵, 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.

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

JoVE 2670 5/23/2011

Vera Brümmer¹, Stefan Schneider¹, Tobias Vogt¹, Heiko Strüder¹, Heather Carnahan², Christopher D. Askew³, Roland Csuhaj⁴

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.

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

JoVE 2261 5/12/2011

Philippe M. D'Onofrio*, Mark M. Magharious*, Paulo D. Koeberle

Department 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.

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

JoVE 2241 5/12/2011

Mark M. Magharious*, Philippe M. D'Onofrio*, Paulo D. Koeberle

Department 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.

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

JoVE 2497 2/27/2011

Jun Dazai*¹, Shoshana Spring*¹, Lindsay S. Cahill¹, 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.

Assessment of Social Interaction Behaviors

JoVE 2473 2/25/2011

Oksana Kaidanovich-Beilin¹, Tatiana Lipina¹, Igor Vukobradovic², 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.

A Protocol for Comprehensive Assessment of Bulbar Dysfunction in Amyotrophic Lateral Sclerosis (ALS)

JoVE 2422 2/21/2011

Yana Yunusova^1,2, Jordan R. Green³, Jun Wang³, Gary Pattee⁴, 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.

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

JoVE 2325 1/18/2011

Martha M. Robinson¹, Jonathan M. Martin¹, Harold L. Atwood², Robin L. Cooper¹

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.

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

JoVE 2322 1/18/2011

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.

An Investigation of the Effects of Sports-related Concussion in Youth Using Functional Magnetic Resonance Imaging and the Head Impact Telemetry System

JoVE 2226 1/12/2011

Michelle Keightley^1,2,3,4,5, Stephanie Green¹, Nick Reed¹, Sabrina Agnihotri¹, Amy Wilkinson³, 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.

Microfabricated Platforms for Mechanically Dynamic Cell Culture

JoVE 2224 12/26/2010

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.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

JoVE 2094 12/19/2010

Usheer Kanjee, Walid A. Houry

Department 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.

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

JoVE 2177 12/13/2010

M. Dean Chamberlain¹, Mark J. Butler¹, Ema C. Ciucurel¹, Lindsay E. Fitzpatrick², Omar F. Khan¹, Brendan M. Leung², Chuen Lo¹, Ritesh Patel², Alexandra Velchinskaya², Derek N. Voice², Michael V. Sefton¹

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.

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

JoVE 2387 12/04/2010

Serge Gueroussov, Stefan P. Tarnawsky, Xianying A. Cui, Kohila Mahadevan, Alexander F. Palazzo

Department 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.

Isolation of Retinal Stem Cells from the Mouse Eye

JoVE 2209 9/11/2010

Brenda L.K. Coles, Derek van der Kooy

Molecular 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.

Eye Movement Monitoring of Memory

JoVE 2108 8/15/2010

Jennifer D. Ryan^1,2,3, Lily Riggs^1,2, Douglas A. McQuiggan¹

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.

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

JoVE 2130 8/13/2010

Ahmad Zaheen, Alberto Martin

Department 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.

Dissection of Oenocytes from Adult Drosophila melanogaster

JoVE 2242 7/18/2010

Joshua J. Krupp, Joel D. Levine

Department 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.

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

JoVE 2050 7/08/2010

Christina Farr¹, 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.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

JoVE 1698 2/01/2010

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.

Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging

JoVE 1581 11/25/2009

Melissa Paulite¹, Zahra Fakhraai², Boris B. Akhremitchev³, Kerstin Mueller¹, Gilbert C. Walker¹

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.

Digital Microfluidics for Automated Proteomic Processing

JoVE 1603 11/06/2009

Mais J. Jebrail¹, Vivienne N. Luk^1,2, Steve C. C. Shih^2,3, Ryan Fobel^2,3, Alphonsus H. C. Ng^2,3, Hao Yang¹, Sergio L. S. Freire¹, 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.