University of Toronto

146 articles published in JoVE

• Immunology and Infection

  
  Scoring Central Nervous System Inflammation, Demyelination, and Axon Injury in Experimental Autoimmune Encephalomyelitis Carmen C. Ucciferri¹, Annette Gower², Nuria Alvarez-Sanchez^1,2, Heather Whetstone³, Valeria Ramaglia¹, Jennifer L. Gommerman¹, Koroboshka Brand-Arzamendi², Raphael Schneider^2,4, Shannon E Dunn^{1,2,5 1}Department of Immunology, University of Toronto, ²Keenan Research Centre for Biomedical Science of St. Michael’s Hospital, ³Sickkids Research Institute, The Hospital for Sick Children, ⁴Women’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.

• 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 1}Latner 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.

• Medicine

  
  Measuring Diaphragm Thickness and Function Using Point-of-Care Ultrasound Catherine A. Bellissimo¹, Idunn S. Morris^2,3,4, Jenna Wong¹, Ewan C. Goligher^{1,2,3,5 1}Toronto 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.

• Neuroscience

  
  Quantification of Visual Feature Selectivity of the Optokinetic Reflex in Mice Jiashu Liu^1,2, Bao-hua Liu^{1,2 1}Department 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.

• Neuroscience

  
  BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABA_A Receptor Presentation in the Rodent Brain Akiko Sumitomo¹, Runhao Zhou^1,2, Thomas Prevot^1,3, Etienne Sibille^1,2,3, Toshifumi Tomoda^{1,3 1}Centre 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.

• 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 Lao¹, Arneet L. Saltzman^{1 1}Department 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.

• 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 Piorkowska³, Ben Keunen³, Yael Babichev², Suzanne M. Wong^3,4, Maximilian Regenold⁵, Michael Dunne⁵, Julia Nomikos^1,2, Maryam Siddiqui⁶, Samuel Pichardo⁶, Warren Foltz⁷, Adam C. Waspe^3,8, Justin T. Gerstle^3,9, James M. Drake^1,3,4,10, Rebecca A. Gladdy^{1,2,10 1}Institute 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.

• 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 1}Laboratory 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.

• Bioengineering

  
  Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor Michael S. Xu¹, Golnaz Karoubi^1,2, Thomas K. Waddell^1,3, Siba Haykal^{1,4 1}Latner 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.

• 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 Paola³, Nicola L. Jones^2,4,5, Christine E. Bear^{1,3,5 1}Molecular 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.

• 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 Norouzi¹, Pouriya Bayat¹, Margot Karlikow¹, Seray Cicek¹, Yuxiu Guo¹, Alexander A. Green^3,4, Lindomar Pena², Keith Pardee^{1,5 1}Leslie 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.

• 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 1}Latner 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.

• 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 1} 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.

• Medicine

  
  Conducting Respiratory Oscillometry in an Outpatient Setting Ehren Chang¹, Anastasiia Vasileva¹, Cynthia Nohra¹, Clodagh M. Ryan¹, Chung-Wai Chow¹, Joyce Ka Yan Wu^{1 1}Division 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.

• Immunology and Infection

  
  Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection Alexandra R. Willis¹, Hala Tamim El Jarkass¹, Aaron W. Reinke^{1 1}Department 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.

• 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 Salvo², Martina Avesani², Jolanda Sabatino², Alvise Guariento^1,3, Vladimiro Vida^{1 1}Pediatric 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.

• 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 Davies³, Kullervo Hynynen^{1,2,6 1}Physical 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.

• 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 1}Department 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.

• Bioengineering

  
  DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation Ryan C. Lee¹, Travis R. Douglas¹, Leo Y. T. Chou^{1 1}Institute 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.

• Medicine

  
  Synthesis and Characterization of Multi-Modal Phase-Change Porphyrin Droplets Kimoon Yoo^1,2, Alexander Dhaliwal^1,2, Juan Chen¹, Paul S. Sheeran³, Gang Zheng^{1,2 1}Princess 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.

• Behavior

  
  Handling Techniques to Reduce Stress in Mice Michael Marcotte¹, Ashley Bernardo^1,2, Nathaniel Linga³, Carmina A. Pérez-Romero⁴, Jean-Louis Guillou⁵, Etienne Sibille^1,2,3, Thomas D. Prevot^{1,2 1}Campbell 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).

• Biology

  
  Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells Magdalena M. Szewczyk¹, Victoria Vu¹, Dalia Barsyte-Lovejoy^{1,2 1}Structural 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.

• Biochemistry

  
  Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System Ashley Hutchinson¹, Almagul Seitova^{1 1}Structural 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.

• Bioengineering

  
  Control of Cell Geometry through Infrared Laser Assisted Micropatterning Shuying Yang¹, Chen Tuo¹, Ernest Iu¹, Sergey V. Plotnikov^{1 1}Department 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.

• 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 Correa¹, Jane Batt^{1,2 1}Keenan 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.

• Biology

  
  Quantitative Analysis of Cell Edge Dynamics during Cell Spreading Ernest Iu*¹, Alexander Bogatch*¹, Sergey V. Plotnikov^{1 1}Department 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.

• 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 1}Program 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.

• 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 1}Institute 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.

• 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 1}Krembil 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.

• 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 1}Department 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.

• Bioengineering

  
  Capturing Representative Hand Use at Home Using Egocentric Video in Individuals with Upper Limb Impairment Meng-Fen Tsai^1,3, Andrea Bandini³, Rosalie H. Wang^2,3,5, José Zariffa^{1,3,4,5 1}Institute 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.

• 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 Manuel¹, Sai Chung^1,2, Eddie He¹, Agata Bartczak¹, Sandra Fischer³, Cornelia Thoeni³, Graziano Oldani¹, Catia T. Perciani¹, Sonya MacParland^1,2,3, Ian McGilvray^{1 1}Multi-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.

• Genetics

  
  In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity Sampath Kumar Loganathan¹, Ahmad Malik^1,2, Ellen Langille^1,2, Chen Luxenburg³, Daniel Schramek^{1,2 1}Centre 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.

• Bioengineering

  
  Blood Flow Imaging with Ultrafast Doppler Jerome Baranger^1,2, Luc Mertens^1,2, Olivier Villemain^{1,2,3 1}Translational 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).

• Biology

  
  Quantification of Circulating Pig-Specific DNA in the Blood of a Xenotransplantation Model Yangyang Deng*^1,2, Ming Zhou*^1,3, Ying Lu¹, Jiao Chen¹, Zuhui Pu⁴, Dongjing Yu^1,5, Yifan Dai⁶, Yongqiang Zhan⁷, Lisha Mou^{1 1} 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.

• 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 1}Department 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.

• 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 Padmanabhan², Marco A.S. Baptista², Lindsey Riley², Danielle Smith³, Connie Marras⁴, Andrew Howden⁵, Dario R. Alessi¹, Esther Sammler^{1,6 1}MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, ² 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.

• 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 1}Department 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.

• 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 1}University 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.

• Cancer Research

  
  An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells Lauren Philp^1,2, Harley Chan³, Marjan Rouzbahman⁴, Ariana Rostami⁵, Lili Ding⁶, Scott V. Bratman^5,6, Margarete K. Akens^5,7,8, Jonathan C. Irish^7,9,11, Marcus Q. Bernardini¹⁰, Gang Zheng^{5,11 1}Institute 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.

• Genetics

  
  Pooled CRISPR-Based Genetic Screens in Mammalian Cells Katherine Chan*¹, Amy Hin Yan Tong*¹, Kevin R Brown¹, Patricia Mero¹, Jason Moffat^{1,2,3 1}Donnelly 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.

• Medicine

  
  A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation Roberto V. P. Ribeiro^1,2, Juglans S. Alvarez¹, Frank Yu¹, Mitchell B. Adamson^1,2, Naoto Fukunaga¹, Cyril Serrick³, Ved Bissoondath¹, Massimiliano Meineri^4,5, Mitesh V. Badiwala^1,6, Vivek Rao^{1,2,6 1}Division 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.

• Bioengineering

  
  Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles Hisato Konoeda¹, Hong Yang², Chengliang Yang¹, Annette Gower¹, Chun Xu¹, Wei Zhang¹, Mingyao Liu^{1,3 1}Latner 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.

• 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 1}Division 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.

• 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 An¹, Patrick E. Steadman³, Jae-Ryong Kim⁴, Hoon-Ki Sung^{1,2,5 1}Translational 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.

• 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 1}Institute of Medical Science, University of Toronto, ² 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.

• Medicine

  
  Autonomic Function Following Concussion in Youth Athletes: An Exploration of Heart Rate Variability Using 24-hour Recording Methodology Melissa Paniccia¹, Tim Taha², Michelle Keightley^1,3, Scott Thomas², Lee Verweel¹, James Murphy¹, Katherine Wilson¹, Nick Reed^{1,3,4 1}Concussion 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.

• 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 Xu², Cindi M. Morshead^{1,2,3 1}Institute 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.

• 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 1}Toronto 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.

• Neuroscience

  
  Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex Pantelis Lioumis¹, Reza Zomorrodi¹, Itay Hadas¹, Zafiris J. Daskalakis^1,2, Daniel M. Blumberger^{1,2 1}Temerty 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.

• Biochemistry

  
  Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques June Ereño-Orbea*¹, Taylor Sicard*^1,2, Hong Cui¹, Indira Akula¹, Jean-Philippe Julien^{1,2,3 1}Program 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.

• Biology

  
  In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge Oliver A. Kent¹, Charles Steenbergen², Samarjit Das^{2 1}Princess 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.

• Environment

  
  Determination of the Settling Rate of Clay/Cyanobacterial Floccules Tiffany Playter¹, Kurt Konhauser¹, George W. Owttrim², Denise S. Whitford², Tyler Warchola¹, Cheryl Hodgson^1,3, Aleksandra M. Mloszewska⁴, Bruce Sutherland¹, J.-P. Zonneveld¹, S. George Pemberton¹, Murray K. Gingras^{1 1}Department 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.

• 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 Hou¹, Donghui Wu^{1 1}Laboratory 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.

• Medicine

  
  Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae Tali Farine^1,2, Michael Parsons², Stephen Lye^1,2,3, Oksana Shynlova^{1,2,3 1}Department 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.

• Genetics

  
  Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease Allison A. Dilliott^1,2, Sali M.K. Farhan³, Mahdi Ghani⁴, Christine Sato⁴, Eric Liang⁵, Ming Zhang⁴, Adam D. McIntyre¹, Henian Cao¹, Lemuel Racacho^6,7, John F. Robinson¹, Michael J. Strong^1,8, Mario Masellis^9,10, Dennis E. Bulman^6,7, Ekaterina Rogaeva⁴, Anthony Lang^10,11, Carmela Tartaglia^4,10, Elizabeth Finger^12,13, Lorne Zinman⁹, John Turnbull¹⁴, Morris Freedman^10,15, Rick Swartz⁹, Sandra E. Black^9,16, Robert A. Hegele^{1,2 1}Robarts Research Institute, Schulich School of Medicine and Dentistry, Western University, ²Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, ³Analytic and Translational Genetics Unit, Center for Genomic Medicine, Harvard Medical School, Massachusetts General Hospital, Stanley Centre for Psychiatric Research, Broad Institute of MIT and Harvard, ⁴Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, ⁵ Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

• Developmental Biology

  
  Dissection and Staining of Drosophila Pupal Ovaries Karen Sophia Park¹, Dorothea Godt², Daniel Kalderon^{1 1}Department 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.

• Genetics

  
  High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification Allison Jandura^1,2, Jack Hu¹, Ronit Wilk^1,2, Henry M. Krause^{1,2 1}The 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.

• 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 Zhang¹, Tian Zhang¹, King Chen¹, Ji Cheng¹, Paris Lai¹, Andrew M. Rauth², K. Sandy Pang¹, Xiao Yu Wu^{1 1}Department 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.

• Medicine

  
  Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid Shaun Pacheco¹, Shan-Yu Fung², Mingyao Liu^{1,3 1}Latner Thoracic Surgery Research Laboratories, University Health Network, ² 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.

• 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. Gratch¹, Peter Szaraz^1,2, Clifford L. Librach^{1,2,3,4,5 1}Create Fertility Centre, ²Department of Physiology, University of Toronto, ³Department of Obstetrics and Gynecology, University of Toronto, ⁴Department of Medical Sciences, University of Toronto, ⁵ 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.

• Biology

  
  Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets Madina Makhmutova¹, Tao Liang², Herbert Gaisano², Alejandro Caicedo¹, Joana Almaça^{1 1}Department 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.

• Biochemistry

  
  Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy Yulong Sun¹, Philbert Ip², Avijit Chakrabartty^{1,2 1}Department 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.

• 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 1}Create Fertility Centre, ²Department of Physiology, University of Toronto, ³Department of Obstetrics and Gynecology, University of Toronto, ⁴Department of Physiology, University of Toronto, ⁵ 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.

• Bioengineering

  
  Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model Ann Lee-Barthel¹, Keith Baar^1,2, Daniel W. D. West^{1,3 1}Department 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.

• 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. Zheng¹, Bruno Bissonnette^1,2,3, Andrew D. Miller⁴, Tanner L. Koppert^1,2, Joseph D. Tobias^1,2, Christopher R. Pierson^5,6, Fedias L. Christofi^{1 1}Department of Anesthesiology, Ohio State University College of Medicine, ² 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.

• Developmental Biology

  
  Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation Xuetao Sun¹, Sara S. Nunes^{1,2,3,4 1}Toronto 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.

• Medicine

  
  Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents Martin Sidler^1,2,3, Karen J. Aitken¹, Jia Xin Jiang⁴, Darius J. Bägli^{1,2,3 1}Developmental 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.

• 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 1}Toronto 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.

• Engineering

  
  Solvent Bonding for Fabrication of PMMA and COP Microfluidic Devices Alwin M. D. Wan¹, Thomas A. Moore^1,2, Edmond W. K. Young^{1,2 1}Department 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.

• Developmental Biology

  
  Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach Yun-Ui Bae¹, Hoon-Ki Sung^2,3, Jae-Ryong Kim^{1 1}Department 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.

• Immunology and Infection

  
  Use of a Monocyte Monolayer Assay to Evaluate Fcγ Receptor-mediated Phagocytosis Tik Nga Tong¹, Donald R. Branch^{1,2 1}Department 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.

• Immunology and Infection

  
  Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model Trevor Beaudoin¹, Sarah Kennedy², Yvonne Yau³, Valerie Waters^{4 1}Physiology 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.

• 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 Mallevaey¹, Shannon E. Dunn^{1,2,3 1}Department of Immunology, University of Toronto, ²Toronto General Research Institute, University Health Network, ³ 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.

• 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 Kagoya², Mark Anczurowski^1,2, Chung-Hsi Wang^1,2, Muhammed A. Rahman², Kayoko Saso², Marcus O. Butler^1,2, Naoto Hirano^{1,2 1}Department 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.

• Chemistry

  
  Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by π-π Stacking Interactions Frantz Le Dévédec¹, Loujin Houdaihed¹, Christine Allen^{1 1}Leslie 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.

• 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 1}Institute 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.

• Immunology and Infection

  
  Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies Andrzej Chruscinski¹, Flora Y. Y. Huang¹, Antigona Ulndreaj², Conan Chua¹, Michael Fehlings³, Vivek Rao⁴, Heather J. Ross¹, Gary A. Levy^{1 1}Multi-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.

• Medicine

  
  Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors Shawn Stapleton^1,2,3, Daniel Mirmilshteyn², Jinzi Zheng^3,4, Christine Allen^2,4,5, David A. Jaffray^{1,2,3,4,5,6 1}Department 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.

• Bioengineering

  
  A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli Matthew J. Leineweber¹, Sam Shi², Jan Andrysek^{1,2 1}Bloorview 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.

• 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 Bu¹, Ralph DaCosta^{1,2,3 1}Princess 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.

• Medicine

  
  Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model Lee Chin^1,2, Elina Korpela³, Anthony Kim¹, Darren Yohan², Carolyn Niu⁴, Brian C. Wilson³, Stanley K. Liu^{1,3 1}Department 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.

• Neuroscience

  
  Foraging Path-length Protocol for Drosophila melanogaster Larvae Ina Anreiter¹, Oscar E. Vasquez¹, Aaron M. Allen², Marla B. Sokolowski^{1,3 1}Department 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.

• Developmental Biology

  
  Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells Sakthi D. Moorthy¹, Jennifer A. Mitchell^{1 1}Department 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).

• Neuroscience

  
  High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem Larissa McKetton¹, Joy Williams², Joseph D. Viviano¹, Yeni H. Yücel³, Neeru Gupta³, Keith A. Schneider^{1 1}Department 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.

• Bioengineering

  
  Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance Yannan N. Dou¹, Robert A. Weersink^2,5, Warren D. Foltz^5,6, Jinzi Zheng^5,6, Naz Chaudary⁷, David A. Jaffray^2,3,4,5,6, Christine Allen^{1 1}Leslie 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.

• Developmental Biology

  
  Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye Sarah J. Smith*^1,2, Eric J. Horstick*^3,4, Ann E. Davidson^1,2, James Dowling^{1,2,4 1}Program in Genetics & Genome Biology, The Hospital for Sick Children, ²Department of Molecular Genetics, The University of Toronto, ³Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, ⁴Departments of Pediatrics and Neurology, University of Michigan In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

• Neuroscience

  
  High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging Julie Winterburn^1,2, Jens C. Pruessner³, Chavez Sofia^4,5, Mark M. Schira^6,7, Nancy J. Lobaugh^4,8, Aristotle N. Voineskos^5,9, M. Mallar Chakravarty^{1,2 1}Institute 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.

• Medicine

  
  In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose Venkatesh Krishnan¹, Robert Clark¹, Marina Chekmareva², Amy Johnson¹, Sophia George³, Patricia Shaw⁴, Victoria Seewaldt^4,5, Carrie Rinker-Schaeffer^{1 1}Section of Urology, Department of Surgery, The University of Chicago, ²Department of Pathology, Robert Wood Johnson Medical School, ³Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University Health Network, ⁴Department of Laboratory Medicine and Pathobiology, Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University of Toronto, University Health Network, ⁵Departments of Medicine, Pharmacology, and Cancer Biology, Duke University Medical Center We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

• Biology

  
  Identification of Kinase-substrate Pairs Using High Throughput Screening Courtney Reeks¹, Robert A. Screaton^{2,3 1} 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.

• Medicine

  
  MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder Katharine Dunlop¹, Pauline Gaprielian⁶, 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 1}Institute 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.

• Chemistry

  
  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. Brenan¹, Yingwei Fei^{2 1}Department 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.

• Medicine

  
  Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury Arnaud Dubory^1,2, Elisabeth Laemmel¹, Anna Badner³, Jacques Duranteau^1,4, Eric Vicaut¹, Charles Court², Marc Soubeyrand^{1,2 1}Laboratoire 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.

• Behavior

  
  Testing Sensory and Multisensory Function in Children with Autism Spectrum Disorder Sarah H. Baum¹, Ryan A. Stevenson², Mark T. Wallace^{3 1}Vanderbilt 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.

• Biology

  
  Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein Paul D. W. Eckford¹, Canhui Li¹, Christine E. Bear^{1,2,3 1}Programme 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.

• 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 1}Department 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.

• Developmental Biology

  
  Organotypic Slice Cultures for Studies of Postnatal Neurogenesis Adam J. Mosa¹, Sabrina Wang^2,3, Yao Fang Tan¹, J. Martin Wojtowicz^{1 1}Department 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.

• Medicine

  
  Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury Klaus Zweckberger¹, Yang Liu¹, Jian Wang¹, Nicole Forgione¹, Michael G. Fehlings^{1,2,3 1}Department 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.

• Immunology and Infection

  
  Electroporation of Functional Bacterial Effectors into Mammalian Cells Ryan L. Sontag¹, Cosmin Mihai², Galya Orr², Alexei Savchenko³, Tatiana Skarina³, Hong Cui³, John R. Cort¹, Joshua N. Adkins¹, Roslyn N. Brown^{4 1}Biological 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.

• 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 1}The 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.

• Neuroscience

  
  Long-term Time Lapse Imaging of Mouse Cochlear Explants Joanna F. Mulvaney¹, Alain Dabdoub^{1,2,3 1}Biological 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.

• Neuroscience

  
  Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun Jason Arsenault^1,2, Andras Nagy¹, Jeffrey T. Henderson¹, John A. O'Brien^{2 1}Leslie 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.

• Immunology and Infection

  
  Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells Martina Kučerová-Levisohn¹, Jordana Lovett¹, Armin Lahiji¹, Roxanne Holmes², Juan Carlos Zúñiga-Pflücker², Benjamin D. Ortiz^{1 1}Department 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.

• Medicine

  
  A Multi-Modal Approach to Assessing Recovery in Youth Athletes Following Concussion Nick Reed^1,2,3, James Murphy¹, Talia Dick¹, Katie Mah³, Melissa Paniccia³, Lee Verweel³, Danielle Dobney³, Michelle Keightley^{1,2,3 1}Bloorview 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.

• Neuroscience

  
  Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient Marie Kristel Bermejo*¹, Marija Milenkovic*¹, Ali Salahpour¹, Amy J. Ramsey^{1 1}Department 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.

• Environment

  
  Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers Devrim Coskun¹, Dev T. Britto¹, Ahmed M. Hamam¹, Herbert J. Kronzucker^{1 1}Department 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.

• Biology

  
  Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands Rushin Shojaii¹, Stephanie Bacopulos^2,3, Wenyi Yang^2,3, Tigran Karavardanyan⁴, Demetri Spyropoulos⁵, Afshin Raouf⁶, Anne Martel^1,4, Arun Seth^{2,3 1}Department 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.

• Medicine

  
  Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly Joel Ramirez¹, Christopher J.M. Scott¹, Alicia A. McNeely¹, Courtney Berezuk¹, Fuqiang Gao¹, Gregory M. Szilagyi^1,2, Sandra E. Black^{1,2 1}LC 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.

• Bioengineering

  
  An Improved Mechanical Testing Method to Assess Bone-implant Anchorage Spencer Bell¹, Elnaz Ajami¹, John E. Davies^{1 1}Institute 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.

• Bioengineering

  
  One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles Cheng S. Jin^1,2,3, Jonathan F. Lovell⁴, Gang Zheng^{1,2,3 1}Department 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.

• Chemistry

  
  Template Directed Synthesis of Plasmonic Gold Nanotubes with Tunable IR Absorbance Colin R. Bridges¹, Tyler B. Schon¹, Paul M. DiCarmine¹, Dwight S. Seferos^{1 1}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.

• Engineering

  
  Bringing the Visible Universe into Focus with Robo-AO 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 Punnadi^{4 1}Caltech 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.

• Biology

  
  Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells Xianying A. Cui¹, Alexander F. Palazzo^{1 1}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.

• Biology

  
  The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions Yo Suzuki¹, Jason Stam¹, Mark Novotny², Nozomu Yachie³, Roger S. Lasken², Frederick P. Roth^{3,4 1}Department 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.

• Neuroscience

  
  C. elegans Tracking and Behavioral Measurement Jirapat Likitlersuang¹, Greg Stephens^2,3, Konstantine Palanski¹, William S. Ryu^{1,4 1}Donnelly 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.

• Biology

  
  Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry Mohan Babu^1,2, Olga Kagan¹, Hongbo Guo¹, Jack Greenblatt^1,3, Andrew Emili^{1,3 1}Banting 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.

• 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 1}Department 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.

• Neuroscience

  
  A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field Robart Babona-Pilipos¹, Milos R. Popovic², Cindi M. Morshead^{3 1}Institute 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.

• 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. Cheng¹, Ning Su¹, M. Cynthia Goh^{1,2 1}Department 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.

• 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 1}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.

• Immunology and Infection

  
  Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation Rahul Kushwah¹, Jim Hu^{2 1}Stem 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.

• Medicine

  
  MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR) Aida Gordanpour¹, Robert K. Nam², Linda Sugar³, Stephanie Bacopulos¹, Arun Seth^{1,3,4 1}Department 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.

• 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-Mata³, Katalin Szaszi^{1,2 1}Keenan 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.

• Medicine

  
  MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model Meaghan A. O'Reilly¹, Adam C. Waspe^1,2, Rajiv Chopra^1,2, Kullervo Hynynen^{1,3 1}Imaging 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.

• 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 Proctor⁵, Lawrence E. Heisler^2,3, Guri Giaever^2,6, Corey Nislow^{1,2,3 1}Banting 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.

• Neuroscience

  
  Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions Vera Brümmer¹, Stefan Schneider¹, Tobias Vogt¹, Heiko Strüder¹, Heather Carnahan², Christopher D. Askew³, Roland Csuhaj^{4 1}Institute 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.

• 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 1}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.

• 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 1}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.

• Neuroscience

  
  Multiple-mouse Neuroanatomical Magnetic Resonance Imaging Jun Dazai*¹, Shoshana Spring*¹, Lindsay S. Cahill¹, R. Mark Henkelman^{1,2 1}Mouse 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.

• Neuroscience

  
  Assessment of Social Interaction Behaviors Oksana Kaidanovich-Beilin¹, Tatiana Lipina¹, Igor Vukobradovic², John Roder^1,3,4,5, James R. Woodgett^{1,3 1}Samuel 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.

• Medicine

  
  A Protocol for Comprehensive Assessment of Bulbar Dysfunction in Amyotrophic Lateral Sclerosis (ALS) Yana Yunusova^1,2, Jordan R. Green³, Jun Wang³, Gary Pattee⁴, Lorne Zinman^{2,5 1}Department 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.

• Biology

  
  Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises Martha M. Robinson¹, Jonathan M. Martin¹, Harold L. Atwood², Robin L. Cooper^{1 1}Department 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.

• 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 1}Department 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.

• 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 Green¹, Nick Reed¹, Sabrina Agnihotri¹, Amy Wilkinson³, Nancy Lobaugh^{6,7 1}Graduate 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.

• Bioengineering

  
  Microfabricated Platforms for Mechanically Dynamic Cell Culture Christopher Moraes^1,2, Yu Sun^1,2, Craig A. Simmons^{1,2,3 1}Department 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.

• Biology

  
  An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase Usheer Kanjee¹, Walid A. Houry^{1 1}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.

• Bioengineering

  
  Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells 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. Sefton^{1 1}Institute 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.

• Biology

  
  Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection Serge Gueroussov¹, Stefan P. Tarnawsky¹, Xianying A. Cui¹, Kohila Mahadevan¹, Alexander F. Palazzo^{1 1}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.

• Biology

  
  Isolation of Retinal Stem Cells from the Mouse Eye Brenda L.K. Coles¹, Derek van der Kooy^{1 1}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.

• Neuroscience

  
  Eye Movement Monitoring of Memory Jennifer D. Ryan^1,2,3, Lily Riggs^1,2, Douglas A. McQuiggan^{1 1}Rotman 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.

• Immunology and Infection

  
  Induction and Assessment of Class Switch Recombination in Purified Murine B Cells Ahmad Zaheen¹, Alberto Martin^{1 1}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.

• Biology

  
  Dissection of Oenocytes from Adult Drosophila melanogaster Joshua J. Krupp¹, Joel D. Levine^{1 1}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.

• Immunology and Infection

  
  Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry Christina Farr¹, Stuart Berger^{1,2 1}Immunology, 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.

• 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 1}Department 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.

• Biology

  
  Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging Melissa Paulite¹, Zahra Fakhraai², Boris B. Akhremitchev³, Kerstin Mueller¹, Gilbert C. Walker^{1 1}Department 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.

• Biology

  
  Digital Microfluidics for Automated Proteomic Processing 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 1}Department 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.