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University of Calgary

51 ARTICLES PUBLISHED IN JoVE

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Biology

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain
John Jarrell 1
1Department of Obstetrics and Gynecology, Faculty of Medicine, University of Calgary

A demonstration of the bedside test for cutaneous allodynia and its clinical implications.

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Neuroscience

Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats
Aubrey A. Webb 1,2, Brendan Kerr 3, Tanya Neville 3, Sybil Ngan 4, Hisham Assem 3
1CullenWebb Animal Neurology & Ophthalmology Center, Riverview, NB, 2Department of Clinical Neurosciences, Faculty of Medicine, University of Calgary, 3Department of Comparative Biology and Experimental Medicine, University of Calgary, 4Department of Neuroscience, University of Calgary

Locomotion is often examined as a behavioural outcome in various models of disease in fields such as neuroscience and orthopedics. This video paper intends to describe a method for collecting ground reaction forces and kinematics from rats during unrestrained locomotion.

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Biology

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers
Curtis C. Hughey 1, Dustin S. Hittel 1,2, Virginia L. Johnsen 2, Jane Shearer 1,2
1Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 2Faculty of Kinesiology, University of Calgary

Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.

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Medicine

Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat
Curtis C. Hughey 1, Dustin S. Hittel 1,2, Virginia L. Johnsen 2, Jane Shearer 1,2
1Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 2Faculty of Kinesiology, University of Calgary

The hyperinsulinemic-euglycemic clamp is the "gold standard" for the assessment of insulin action. Insulin is infused at a constant rate stimulating glucose uptake. The amount of exogenous glucose infused to counter this drop is indicative of insulin sensitivity. Here the procedure is performed on a conscious, unrestrained rat.

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Biology

Germ Cell Transplantation and Testis Tissue Xenografting in Mice
Lin Tang *1, Jose Rafael Rodriguez-Sosa *1, Ina Dobrinski 1
1Department of Comparative Biology and Experimental Medicine, University of Calgary

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

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Immunology and Infection

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions
Anutosh Ganguly 1, Hong Zhang 1, Ritu Sharma 1, Sean Parsons 1, Kamala D. Patel 1
1Department of Physiology and Pharmacology, University of Calgary

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

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Bioengineering

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates
Golsa Razian 1, Yang Yu 1, Mark Ungrin 1
1Department of Comparative Biology & Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

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Neuroscience

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining
Richelle Mychasiuk 1, Robbin Gibb 1, Bryan Kolb 1
1Canadian Centre for Behavioural Neuroscience, University of Lethbridge

This paper describes the procedures for tactile stimulation of rat pups and subsequent Golgi-Cox staining of neuronal morphology. Tactile stimulation is a positive experience that is administered in the perinatal period by stroking pups with a household duster. Golgi-cox staining is a reliable procedure permitting the visualization of entire neurons.

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Immunology and Infection

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus
Jo-Ann McClure-Warnier 1, John M. Conly 1,2,3,4,5, Kunyan Zhang 1,2,3,4,5
1Centre for Antimicrobial Resistance, Alberta Health Services / Calgary Laboratory Services / University of Calgary, 2Department of Pathology & Laboratory Medicine, University of Calgary, 3Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, 4Department of Medicine, University of Calgary, 5The Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, University of Calgary

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

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Behavior

Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism
Jacklyn D. Smith 1, Jong M. Rho 1, Susan A. Masino 2, Richelle Mychasiuk 1
1Departments of Paediatrics and Clinical Neurosciences, Alberta Children's Hospital Research Institute, University of Calgary Faculty of Medicine, 2Department of Psychology, Neuroscience Program, Trinity College

Inchworming is a highly repetitive synchronous digging motion displayed by BTBR T+ Itpr3tf/J (BTBR) mice when placed in a testing cage with sufficient sawdust bedding. The procedure is a modification of the juvenile social interaction protocol and is used here to assess repetitive motor stereotypies relevant to Autism Spectrum Disorder.

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Chemistry

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence
Zachary J. Storms 1, Elliot Cameron 1, Hector de la Hoz Siegler 1,2, William C. McCaffrey 1
1Department of Chemical and Materials Engineering, University of Alberta, 2Department of Chemical and Petroleum Engineering, University of Calgary

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

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Bioengineering

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology
Dominique Tremblay 1, Charles M. Cuerrier 1,2, Lukasz Andrzejewski 1, Edward R. O'Brien 3, Andrew E. Pelling 1,4,5
1Centre for Interdisciplinary NanoPhysics, Department of Physics, University of Ottawa, 2University of Ottawa Heart Institue, University of Ottawa, 3Libin Cardiovascular Institute of Alberta, University of Calgary, 4Department of Biology, University of Ottawa, 5Institute for Science, Society and Policy, University of Ottawa

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

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Medicine

A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats
Richelle Mychasiuk 1, Allyson Farran 1, Mariana Angoa-Perez 2, Denise Briggs 2, Donald Kuhn 2, Michael J. Esser 1
1Alberta Children's Hospital Research Institute, University of Calgary, 2Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine & John D. Dingell VA Medical Center

The modified weight-drop technique is an easy, cost-effective procedure used for the induction of mild traumatic brain injury in juvenile rats. This novel technique produces clinically relevant symptomology that will advance the study of mild traumatic brain injury (mTBI) and concussion.

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Behavior

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

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

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Neuroscience

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time
Starlyn L. M. Okada 1, Nicole S. Stivers 1, Peter K. Stys 2, David P. Stirling 1
1KY Spinal Cord Injury Research Center, Department of Neurological Surgery, University of Louisville, 2Hotchkiss Brain Institute, Department of Clinical Neurosciences, University of Calgary

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

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Sequential Radial probe Endobronchial Ultrasound and Electromagnetic Navigation Bronchoscopy for the Diagnosis of Peripheral Pulmonary Lesions
Alex ChunMin Chee 1, Paul MacEachern 1, Christopher Hergott 1, Elaine Dumoulin 1, Alain Tremblay 1
1Division of Respirology, Department of Medicine, University of Calgary

This protocol describes a technique for bronchoscopy using radial probe endobronchial ultrasound to localize and sample peripheral lung lesions with electromagnetic navigation bronchoscopy used as a navigation tool if needed.

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Immunology and Infection

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection
Constance Finney 1, Lena Serghides 2
1Department of Biological Sciences, University of Calgary, 2Toronto General Research Institute, University Health Network

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

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Biology

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit
Heather A. Paul 1, Megan C. Hallam 2, Raylene A. Reimer 1,2
1Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 2Faculty of Kinesiology, University of Calgary

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

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Developmental Biology

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

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

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Immunology and Infection

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material
Yo Ching Cheng *1, Samia Hannaoui *1, Theodore Ralph John 2, Sandor Dudas 3, Stefanie Czub 3, Sabine Gilch 1
1Dept. of Ecosystem and Public Health, Calgary Prion Research Units, Faculty of Veterinary Medicine, University of Calgary, 2Dept. of Molecular Biology, University of Wyoming, 3Canadian Food Inspection Agency, Lethbridge Laboratories

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

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Cancer Research

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy
Kristina J. Allan 1,2, Douglas J. Mahoney 1,4, Stephen D. Baird 1, Charles A. Lefebvre 1, David F. Stojdl 1,2,3
1Children's Hospital of Eastern Ontario (CHEO) Research Institute, 2Department of Biology, Microbiology and Immunology, University of Ottawa, 3Department of Pediatrics, University of Ottawa, 4Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

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Medicine

A Saline/Bipolar Radiofrequency Energy Device As an Adjunct for Hemostasis in Solid Organ Injury/Trauma
Thomas W. Clements 1, Chad G. Ball 1
1Department of Surgery, University of Calgary

The goal of this publication is to demonstrate the potential application of a novel device using simulated solid organ injuries in a porcine model.

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Bioengineering

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System
Candice C. Poon 1, Vincent Ebacher 2, Katherine Liu 1, Voon Wee Yong *1,3, John James Patrick Kelly *1
1Department of Clinical Neurosciences, Arnie Charbonneau Cancer Institute, University of Calgary, 2Department of Cell Biology and Anatomy, Hotchkiss Brain Institute, University of Calgary, 3Department of Oncology, Hotchkiss Brain Institute, University of Calgary

Here we describe a protocol for the automated segmentation of fluorescently labeled tissues on slides using a widefield high-content analysis system (WHCAS). This protocol has wide-ranging applications in any field which involves the quantitation of fluorescent markers in biological tissues including the biological sciences, medical engineering, and health sciences.

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Biochemistry

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs
Joseph A. Ross 1, Nehal Thakor 1,2,3
1Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 2Department of Neuroscience and the Canadian Centre for Behavioral Neuroscience (CCBN), University of Lethbridge, 3Arnie Charbonneau Cancer Institute, University of Calgary

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

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Cancer Research

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion
Ian Restall 1, Danielle Anne Bozek 1, Charles Chesnelong 1,2, Samuel Weiss 1, H. Artee Luchman 1
1Hotchkiss Brain Institute, Department of Cell Biology and Anatomy, Cumming School of Medicine, University of Calgary, 2Department of Developmental and Stem Cell Biology Program, Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

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Behavior

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies
Stefanie Gierszewski 1, Derek Baker 1,2, Klaus Müller 3, Jan-Marco Hütwohl 3, Klaus-Dieter Kuhnert 3, Klaudia Witte 1
1Research Group of Ecology and Behavioral Biology, Institute of Biology, University of Siegen, 2University of Calgary, 3Institute of Real-Time Learning Systems, University of Siegen

Using the novel FishSim Animation Toolchain, we present a protocol for non-invasive visual manipulation of public information in the context of mate-choice copying in sailfin mollies. FishSim Animation Toolchain provides an easy-to-use framework for the design, animation and presentation of computer-animated fish stimuli for behavioral experiments with live test fish.

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Biochemistry

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering
Tyler Mrozowich 1, Steffane McLennan 2, Michael Overduin 2,4, Trushar R. Patel 1,3,4
1Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, 2Department of Biochemistry, University of Alberta, 3Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, 4DiscoveryLab, Faculty of Medicine & Dentistry, University of Alberta

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

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Biology

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues
Jo Anne Stratton *1,2,3, Sarthak Sinha *2, Wisoo Shin 2, Elodie Labit 2, Tak-Ho Chu 1,4, Prajay T. Shah 2, Rajiv Midha 1,4, Jeff Biernaskie 1,2,3
1Hotchkiss Brain Institute, University of Calgary, 2Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, 3Alberta Children's Hospital Research Institute, University of Calgary, 4Department of Clinical Neurosciences, Cumming School of Medicine, University of Calgary

This protocol describes the general processes and quality control checks necessary for preparing healthy adult mammalian single cells for droplet-based, high throughput single cell RNA-Seq preparations. Sequencing parameters, read alignment, and downstream single-cell bioinformatic analysis are also provided.

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Medicine

Protocol and Guidelines for Point-of-Care Lung Ultrasound in Diagnosing Neonatal Pulmonary Diseases Based on International Expert Consensus
Jing Liu 1,2, Roberto Copetti 3, Erich Sorantin 4, Jovan Lovrenski 5, Javier Rodriguez-Fanjul 6, Dalibor Kurepa 7, Xing Feng 8, Luigi Cattaross 9, Huayan Zhang 10,11, Misun Hwang 12, Tsu F. Yeh 13,14, Yisrael Lipener 7, Abhay Lodha 15, Jia-Qin Wang 16, Hai-Ying Cao 2,17, Cai-Bao Hu 2,18, Guo-Rong Lyu 19, Xin-Ru Qiu 1,2, Li-Qun Jia 20, Xiao-Man Wang 20, Xiao-Ling Ren 1,2, Jiu-Ye Guo 1,2, Yue-Qiao Gao 1,2, Jian-Jun Li 1,2, Ying Liu 1,2, Wei Fu 1,2, Yan Wang 21, Zu-Lin Lu 1,2, Hua-Wei Wang 8, Li-Li Shang 22
1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The Neonatal Lung Ultrasound Training Base, Chinese College of Critical Ultrasound, 3Emergency Department, Cattinara University Hospital, 4Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 5Faculty of Medicine, University of Novi Sad, Radiology Department, Institute for Children and Adolescents Health Care of Vojvodina, 6Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 7Division of Neonatal-Perinatal Medicine, Cohen Children's Medical Center, 8Department of Neonatology, Children's Hospital of Soochow University, 9Department of Neonatology, Udine University Hospital, 10Center for Newborn Care, Guangzhou Women and Children's Medical Center, 11Division of Neonatology, Children's Hospital of Philadelphia, 12Department of Radiology, Children's Hospital of Philadelphia, 13Division of Neonatology and NICU, Cook County Children's Hospital, University of Illinois, 14Division of Neonatology, Department of Pediatrics, Taipei Medical University, 15Department of Pediatrics and Community Health Sciences, University of Calgary, 16Department of Pediatrics, The Third Affiliated Hospital of Xinxiang Medical University, 17Department of Ultrasound, GE Healthcare, 18Intensive Care Unit, Zhejiang Hospital, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Ultrasound, Beijing Children's Hospital Affiliated with Capital Medical University, 21Department of Neonatology and NICU, Tai'an City Central Hospital of Shandong Province, 22Department of Intensive Care Unit, The Second Affiliated Hospital of Heilongjiang University of Chinese Medicine

Lung ultrasound is a noninvasive and valuable tool for bedside evaluation of neonatal lung diseases. However, a relative lack of reference standards, protocols and guidelines may limit its application. Here, we aim to develop a standardized neonatal lung ultrasound diagnostic protocol to be used in clinical decision-making.

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Medicine

Isolation of Atrial Myocytes from Adult Mice
Hailey J. Jansen 1, Robert A. Rose 1
1Libin Cardiovascular Institute of Alberta, Department of Cardiac Sciences, Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

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Neuroscience

Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain
Adrianna Giuffre 1,2, Lauran Cole 1,2,3, Hsing-Ching Kuo 4,5, Helen L. Carlson 2,4,5, Jeff Grab 6, Adam Kirton 2,3,4,5,7, Ephrem Zewdie 3,4,5
1Department of Neurosciences, University of Calgary, 2Hotchkiss Brain Institute, University of Calgary, 3Cumming School of Medicine, University of Calgary, 4Department of Pediatrics, University of Calgary, 5Alberta Children's Hospital Research Institute, University of Calgary, 6Faculty of Medicine and Dentistry, University of Alberta, 7Department of Clinical Neurosciences, University of Calgary

We demonstrate protocols for the modulation (tDCS, HD-tDCS) and mapping (robotic TMS) of the motor cortex in children.

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Neuroscience

Using Neuron Spiking Activity to Trigger Closed-Loop Stimuli in Neurophysiological Experiments
Leonardo A. Molina 1,2, Victorita E. Ivan 1, Aaron J. Gruber *1, Artur Luczak *1
1Canadian Center for Behavioural Neuroscience, Department of Neuroscience, University of Lethbridge, 2Clinical Neurosciences, Cumming School of Medicine, University of Calgary

This protocol demonstrates how to use an electrophysiological system for closed-loop stimulation triggered by neuronal activity patterns. Sample Matlab code that can be easily modified for different stimulation devices is also provided.

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Medicine

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture
Sadman Sakib 1, Yang Yu 3,4, Anna Voigt 2, Mark Ungrin 2,3,4, Ina Dobrinski 1,2
1Biochemistry and Molecular Biology, University of Calgary, 2Comparative Biology and Experimental Medicine, University of Calgary, 3Biomedical Engineering Graduate Program, University of Calgary, 4Alberta Diabetes Institute, University of Alberta

Here, we present a protocol for the reproducible generation of porcine testicular organoids with testis specific tissue architecture using the commercially available microwell culture system.

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Immunology and Infection

Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy
Laetitia Duler 1, Nghi Nguyen 2, Eric Ontiveros 3, Ronald H. L. Li 2
1Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California, Davis, 2Department of Veterinary Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, 3Department of Veterinary Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis

We describe a method to identify neutrophil extracellular traps (NETs) in formaldehyde-fixed and paraffin-embedded feline cardiogenic arterial thrombi using heat-induced antigen retrieval and a double immunolabeling protocol.

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Immunology and Infection

Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice
Yujia Lin 1,2, Jingna Xue 2, Shan Liao 2
1Department of General Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin Medical University, 2Inflammation Research Network, Department of Microbiology, Immunology and Infectious Diseases, Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary

A protocol to block lymph flow by surgical suturing of afferent lymphatic vessels is presented.

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Medicine

A Novel Right Ventricular Volume and Pressure Loaded Piglet Heart Model for the Study of Tricuspid Valve Function.
Lily Q. Lin 1, Sanaz Hatami 2, James Yashu Coe 1, Timothy M. Colen 1, Consolato Sergi 3, Richard Thompson 4, Elena S. Di Martino 5, Walter Herzog 6, Ziad Abu Sara 6, Darren H. Freed 2, Nee Scze Khoo 1
1Division of Pediatric Cardiology, Department of Pediatrics, University of Alberta, 2Department of Surgery, University of Alberta, 3Department of Laboratory Medicine and Pathology, University of Alberta, 4Department of Biomedical Engineering, University of Alberta, 5Department of Civil Engineering, University of Calgary, 6Faculty of Kinesiology, University of Calgary

A novel recovery piglet heart model with combined pressure and volume overload on the right ventricle is described for the study of tricuspid valve function.

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Cancer Research

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model
Konstantin Stoletov 1, Lian Willetts 2, Perrin H. Beatty 1, John D. Lewis 1
1Department of Oncology, University of Alberta, Edmonton, Alberta, Canada, 2Department of Cell Biology & Anatomy, Cumming School of Medicine, University of Calgary

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

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Neuroscience

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling
Shreeya Kedia 1,3, Sarah L. Erickson 2,3, Guang Yang 1,2,3
1Department of Biochemistry and Molecular Biology; Cumming School of Medicine, University of Calgary, 2Department of Medical Genetics, Cumming School of Medicine, University of Calgary, 3Alberta Children's Hospital Research Institute, University of Calgary

The development of the mammalian brain requires proper control of gene expression at the level of translation. Here, we describe a polysome profiling system with an easy-to-assemble sucrose gradient-making and fractionation platform to assess the translational status of mRNAs in the developing brain.

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Medicine

Neuronavigation and Laparoscopy Guided Ventriculoperitoneal Shunt Insertion for the Treatment of Hydrocephalus
Albert M. Isaacs 1, Chad G. Ball 2, Mark G. Hamilton 1,3
1Division of Neurosurgery, University of Calgary, 2Department of Surgery, University of Calgary, 3Adult Hydrocephalus Program, Department of Clinical Neuroscience, University of Calgary

Patient outcomes of ventriculoperitoneal (VP) shunt surgery, the mainstay treatment for hydrocephalus in adults, are poor due to high shunt failure rates. We present intraoperative footage of VP shunt insertion using neuronavigation and laparoscopy guidance, with the goal to reduce the risks of proximal and distal shunt catheter failures, respectively.

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Medicine

An Intact Pericardium Ischemic Rodent Model
Ali Fatehi Hassanabad 1, Darrell D. Belke 1, Jeannine Turnbull 1, Jameson A. Dundas 1, Vishnu Vasanthan 1, Guoqi Teng 1, Paul W. M. Fedak 1, Justin F. Deniset 1,2
1Section of Cardiac Surgery, Department of Cardiac Sciences, Libin Cardiovascular Institute, Cumming School of Medicine, 2Department of Pharmacology and Physiology, University of Calgary

This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents.

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Immunology and Infection

Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy
Liam Wilkinson 1,2, Johnathan Canton 2,3
1Department of Biochemistry & Microbiology, Faculty of Science, University of Victoria, 2Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Science, University of Calgary, 3Calvin, Joan and Phoebe Snyder Institute for Chronic Diseases, University of Calgary

We describe protocols for measuring pH, oxidative events, and protein digestion in individual macropinosomes in live cells. An emphasis is placed on dual-fluorophore ratiometric microscopy and the advantages it offers over population-based techniques.

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Immunology and Infection

Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings
Yan D. Niu 1, Jae Eun Hyun 1, Nghi Nguyen 1
1Department of Ecosystem and Public Health, Faculty of Veterinary Medicine, University of Calgary

This protocol describes a robust method for using high-throughput settings to screen the antibacterial efficacy of bacteriophage cocktails.

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Biology

Operation of Laboratory Photobioreactors with Online Growth Measurements and Customizable Light Regimes
Marianne Haines 1, Marc Strous 1
1Department of Geoscience, University of Calgary

This publication describes the design of laboratory photobioreactors (PBRs) with customizable light regimes. The growth of cyanobacteria or microalgae, using bicarbonate as their carbon source, is monitored continuously by measuring volumetric oxygen production. These PBRs facilitate rapid, replicated laboratory growth comparisons with little user intervention during experiments.

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Medicine

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses
Kittiwoot To-on 1, Usana Chatturong 1, Teerapap Panklai 2, Iyapa Palang 1, Anjaree Inchan 1, Sutthinee Wisutthathum 3, Tamkeen Urooj Paracha 4, Phapada Apaikawee 1, Krongkarn Chootip 1
1Department of Physiology, Faculty of Medical Science and Center of Excellence for Innovation in Chemistry, Naresuan University, 2Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences, Naresuan University, 3Cosmetics and Natural Products Research Center, Faculty of Pharmaceutical Sciences, Naresuan University, 4Snyder Institute for Chronic Diseases, Airways Inflammation Research Group, Department of Physiology and Pharmacology, University of Calgary

Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli.

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Immunology and Infection

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System
Stefan Hall 1, Sufyan Faridi 2, Irene Euodia 2,3, Sophie Tanner 4, Andrew Krzysztof Chojnacki 5, Kamala D. Patel 6,7, Juan Zhou 2,4, Christian Lehmann 1,2,4,8
1Department of Physiology and Biophysics, Dalhousie University, 2Department of Microbiology and Immunology, Dalhousie University, 3Department of Neuroscience, Dalhousie University, 4Department of Anesthesia, Pain Management, and Perioperative Medicine, Dalhousie University, 5Live Cell Imaging Center, University of Calgary, 6Department of Physiology and Pharmacology, University of Calgary, 7Department of Biochemistry and Molecular Biology, University of Calgary, 8Department of Pharmacology, Dalhousie University

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

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Cancer Research

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

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

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Neuroscience

Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains
Jimena Barbeito-Andrés 1, Laura Andrini 2, Mariana Vallejo-Azar 1, Sara Seguel 3, Jay Devine 4, Benedikt Hallgrímsson 4, Paula Gonzalez 1
1Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos (ENyS, CONICET-UNAJ-HEC), 2Facultad de Ciencias Médicas, Universidad Nacional de La Plata, 3Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, 4Department of Cell Biology and Anatomy, Cumming School of Medicine, University of Calgary

This study describes the steps for obtaining high-resolution images of neonatal mouse brains by combining micro-computed tomography (micro-CT) and a contrast agent in ex vivo samples. We describe basic morphometric analyses to quantify brain size and shape in these images.

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Medicine

Erosion Identification in Metacarpophalangeal Joints in Rheumatoid Arthritis using High-Resolution Peripheral Quantitative Computed Tomography
Yousif Al-Khoury 1,2, Stephanie Finzel 3, Camille Figueiredo 4, Andrew J. Burghardt 5, Kathryn S. Stok 6, Lai-Shan Tam 7, Isaac Cheng 7, Justin J. Tse 1, Sarah L. Manske 1
1Department of Radiology, McCaig Institute for Bone and Joint Health, University of Calgary, 2Department of Biomedical Engineering, University of Calgary, 3University Medical Center Freiburg, 4Department of Rheumatology, University of São Paulo, 5Department of Radiology and Biomedical Imaging, University of California, San Francisco, 6Department of Biomedical Engineering, University of Melbourne, 7Department of Medicine and Therapeutics, The Chinese University of Hong Kong

Bone erosions are an important pathological feature of rheumatoid arthritis. The purpose of this work is to introduce a training tool to provide users with guidance on identifying pathological cortical breaks on high resolution peripheral quantitative computed tomography images for erosion analysis.

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Medicine

Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses
Julian Wolf 1,2, Teja Chemudupati 1,2, Aarushi Kumar 1,2, Ditte K. Rasmussen 1,2, Karen M. Wai 2, Robert T. Chang 2, Artis A. Montague 2, Peter H. Tang 3,4, Alexander G. Bassuk 5,6,7, Antoine Dufour 8,9, Prithvi Mruthrunjaya 2, Vinit B. Mahajan 1,2,10
1Molecular Surgery Laboratory, Stanford University, 2Department of Ophthalmology, Byers Eye Institute, Stanford University, 3Department of Ophthalmology and Visual Neurosciences, University of Minnesota, 4Retina Consultants of Minnesota, 5Department of Pediatrics, University of Iowa, 6Department of Neurology, University of Iowa, 7The Iowa Neuroscience Institute (INI), University of Iowa, 8Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, 9Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 10Veterans Affairs Palo Alto Health Care System

This protocol presents an integrated biorepository platform for the standardized collection, annotation, and biobanking of high-quality human aqueous humor and vitreous liquid biopsies for molecular downstream analyses, including proteomics, metabolomics, and glycomics.

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Neuroscience

Generating and Analyzing High-Parameter Histology Images with Histoflow Cytometry
Rajiv W. Jain 1,2, David A. Elliott 1, V. Wee Yong 1,2
1Hotchkiss Brain Institute, University of Calgary, 2Department of Clinical Neurosciences, University of Calgary

Described here is a method that can be used to image five or more fluorescent parameters by immunofluorescent microscopy. An analysis pipeline for extracting single cells from these images and conducting single-cell analysis through flow cytometry-like gating strategies is outlined, which can identify cell subsets in tissue sections.

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Neuroscience

Pipeline for Planning and Execution of Transcranial Ultrasound Neuromodulation Experiments in Humans
Terra Fairbanks 1,2, Ali K. Zadeh 1,2, Hrishikesh Raghuram 3, Alan Coreas 3, Shirshak Shrestha 4, Siyun Li 3, G. Bruce Pike 1,2,3, Fady Girgis 1,2, Samuel Pichardo 1,2,3
1Department of Clinical Neurosciences, Cumming School of Medicine, University of Calgary, 2Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, 3Department of Radiology, Cumming School of Medicine, University of Calgary, 4Department of Biomedical Engineering, Schulich School of Engineering, University of Calgary

Transcranial ultrasound stimulation (TUS) is an emerging non-invasive neuromodulation technique that requires careful planning of acoustic and thermal simulations. The methodology describes an image processing and ultrasound simulation pipeline for efficient, user-friendly, streamlined planning for human TUS experimentation.

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