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84 ARTICLES PUBLISHED IN JoVE

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Biology

Catheterization of Intestinal Loops in Ruminants
Richard R. E. Uwiera 1, John P. Kastelic 2, G. Douglas Inglis 2
1Department of Agricultural, Food and Nutritional Science, University of Alberta, 2Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge

We describe a novel surgical method for catheterizing 'intestinal loops' within the ileum of sheep. Once animals have recovered from surgery and have cleared antibiotics and analgesics, multiple treatments can be deposited directly in loops via the catheters.

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Biology

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF
Wen Xiong 1, Yan Gao 1, Xun Cheng 1, Charles Martin 1, Dongmei Wu 1, Shuyuan Yao 1, Min-Ju Kim 2, Yang Liu 1
1Research and Development, Stemgent, 2Product Marketing, Stemgent

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

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Neuroscience

Brain Imaging Investigation of the Neural Correlates of Observing Virtual Social Interactions
Keen Sung 1, Sanda Dolcos 2, Sophie Flor-Henry 3, Crystal Zhou 3, Claudia Gasior 4, Jennifer Argo 5, Florin Dolcos 2,6,7
1Department of Computing Science, University of Alberta, 2Department of Psychology, University of Illinois, 3Centre for Neuroscience, University of Alberta, 4Department of Psychology, University of Alberta, 5Department of Marketing, Business Economics, and Law, University of Alberta, 6Neuroscience Program, University of Illinois at Urbana-Champaign, 7Beckman Institute, University of Illinois at Urbana-Champaign

This article demonstrates an experimental design in which whole-body animated characters are used in conjunction with functional magnetic resonance imaging (fMRI) to investigate the neural correlates of observing virtual social interactions.

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Neuroscience

Brain Imaging Investigation of the Neural Correlates of Emotional Autobiographical Recollection
Ekaterina Denkova 1, Trisha Chakrabarty 1, Sanda Dolcos 1,2, Florin Dolcos 1,2,3,4
1Department of Psychiatry, University of Alberta, Edmonton, 2Psychology Department, University of Illinois, Urbana-Champaign, 3Neuroscience Program, University of Illinois, Urbana-Champaign, 4Beckman Institute for Advanced Science & Technology, University of Illinois, Urbana-Champaign

We present a protocol that allows investigation of the neural correlates of recollecting emotional autobiographical memories, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

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Neuroscience

Brain Imaging Investigation of the Neural Correlates of Emotion Regulation
Sanda Dolcos 1, Keen Sung 2, Ekaterina Denkova 3, Roger A. Dixon 4,5, Florin Dolcos 1,6,7
1Department of Psychology, University of Illinois, Urbana-Champaign, 2Department of Computing Science, University of Alberta, Edmonton, 3Department of Psychiatry, University of Alberta, Edmonton, 4Department of Psychology, University of Alberta, Edmonton, 5Centre for Neuroscience, University of Alberta, Edmonton, 6Neuroscience Program, University of Illinois, Urbana-Champaign, 7Beckman Institute, University of Illinois, Urbana-Champaign

We present a protocol that allows investigation of the neural correlates of deliberate and automatic emotion regulation, using functional magnetic resonance imaging. This protocol can be used in healthy participants, both young and older, as well as in clinical patients.

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Neuroscience

Brain Imaging Investigation of the Memory-Enhancing Effect of Emotion
Andrea Shafer 1, Alexandru Iordan 2, Roberto Cabeza 3, Florin Dolcos 1,4
1Centre for Neuroscience, University of Alberta, 2Neuroscience Program, University of Illinois, Urbana-Champaign, 3Center for Cognitive Neuroscience, Duke University, 4Psychology Department, Neuroscience Program, & Beckman Institute, University of Illinois, Urbana-Champaign

We present a protocol that uses functional magnetic resonance imaging to investigate the neural correlates of the memory-enhancing effect of emotion. This protocol allows identification of brain activity specifically linked to memory-related processing, contrary to more general perceptual processing, and can be used with healthy and clinical populations.

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Neuroscience

Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition
Gloria Wong 1,2, Sanda Dolcos 1,3, Ekaterina Denkova 1, Rajendra Morey 4,5,6, Lihong Wang 4,5, Gregory McCarthy 6,7, Florin Dolcos 1,2,3,8,9
1Department of Psychiatry, University of Alberta, 2Centre for Neuroscience, University of Alberta, 3Department of Psychology, University of Illinois, 4Brain Imaging and Analysis Center, Duke University , 5Department of Psychiatry and Behavioral Sciences, Duke University , 6Mid-Atlantic Mental Illness Research Education and Clinical Center, VA Medical Center, 7Department of Psychology, Yale University, 8Neuroscience Program, University of Illinois, 9Beckman Institute for Advanced Science & Technology, University of Illinois

We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

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Biology

Swimming Performance Assessment in Fishes
Keith B. Tierney 1
1Department of Biological Sciences, University of Alberta

The lives of the majority of fish are predicated on swimming. This protocol describes techniques for capturing a range of swimming modes available to individual and schooling fish, and includes metrics associated with swimming physiology and behaviour.

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Biology

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
Aja M. Rieger 1, Kimberly L. Nelson 1, Jeffrey D. Konowalchuk 1, Daniel R. Barreda 1,2
1Department of Biological Sciences, University of Alberta, 2Department of Agriculture, Food and Nutrition Sciences, University of Alberta

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

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Medicine

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas
Eric B. Jelin 1, Samuel C. Schecter 1, Kelly D. Gonzales 1, Shinjiro Hirose 1, Hanmin Lee 1, Geoffrey A. Machin 2, Larry Rand 3, Vickie A. Feldstein 4
1Division of Pediatric and Fetal Surgery, Department of Surgery, University of California, San Francisco, 2Department of Pathology, University of Alberta, 3Department of Obstretics and Gynecology, University of California, San Francisco, 4Department of Radiology, University of California, San Francisco

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins’ circulations. Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

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Medicine

A Swine Model of Neonatal Asphyxia
Po-Yin Cheung 1, Richdeep S. Gill 2, David L. Bigam 2
1Departments of Pediatrics, Pharmacology and Surgery, University of Alberta, 2Department of Surgery, University of Alberta

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

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Medicine

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis
Xiaoqin Hua 1,2, Tobias Deuse 1,2, Evangelos D. Michelakis 3, Alois Haromy 3, Phil S. Tsao 4, Lars Maegdefessel 4, Reinhold G. Erben 5, Claudia Bergow 5, Boris B. Behnisch 6, Hermann Reichenspurner 1,2, Robert C. Robbins 7, Sonja Schrepfer 1,2,7
1University Heart Center Hamburg, TSI-Lab, Germany, 2Cardiovascular Research Center, University of Hamburg, 3Department of Medicine, Cardiology Division, Pulmonary Hypertension Program, University of Alberta, 4Department of Medicine, Stanford University School of Medicine , 5Department of Biomedical Sciences, Institute of Physiology, Pathophysiology, and Biophysics, University of Veterinary Medicine, Vienna, 6Translumina GmbH, Hechingen, 7Department of Cardiothoracic Surgery, Stanford University School of Medicine

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

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Immunology and Infection

Examination of Thymic Positive and Negative Selection by Flow Cytometry
Qian Hu *1, Stephanie A. Nicol *1, Alexander Y.W. Suen *1, Troy A. Baldwin 1
1Department of Medical Microbiology and Immunology, University of Alberta

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

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Engineering

Synthesis and Operation of Fluorescent-core Microcavities for Refractometric Sensing
Shalon McFarlane 1, C.P.K. Manchee 1, Joshua W. Silverstone 1, Jonathan Veinot 1, Al Meldrum 1
1Department of Physics, University of Alberta

Fluorescent-core microcavity sensors employ a high-index quantum-dot coating in the channel of silica microcapillaries. Changes in the refractive index of fluids pumped into the capillary channel cause shifts in the microcavity fluorescence spectrum that can be used to analyze the channel medium.

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Immunology and Infection

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry
Erick García-García 1, Eileen Uribe-Querol 2, Carlos Rosales 3
1Department of Biological Sciences, University of Alberta, 2División de Estudios de Posgrado e Investigación, Facultad de Odontología, Universidad Nacional Autónoma de México, 3Department of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

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Neuroscience

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells
Birbickram Roy 1, Declan William Ali 1
1Department of Biological Sciences, University of Alberta

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

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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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Biology

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae
Miguel F. Gonzales 1, Teresa Brooks 2, Stefan U. Pukatzki 2, Daniele Provenzano 1,3
1Department of Biological Sciences, University of Texas Brownsville, 2Department of Medical Microbiology and Immunology, University of Alberta, 3Department of Biomedical Sciences, University of Texas Brownsville

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

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Engineering

Ultrahigh Density Array of Vertically Aligned Small-molecular Organic Nanowires on Arbitrary Substrates
Ryan Starko-Bowes 1, Sandipan Pramanik 1
1Electrical and Computer Engineering, University of Alberta

We report a simple method for fabricating an ultrahigh density array of vertically ordered small-molecular organic nanowires. This method allows for synthesis of complex heterostructured hybrid nanowire geometries, which can be inexpensively grown on arbitrary substrates. These structures have potential applications in organic electronics, optoelectronics, chemical sensing, photovoltaics and spintronics.

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Medicine

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies
Mukti R. Parikh 1, Andrew R. Belch 2,3, Linda M Pilarski 2,3, Julia Kirshner 1
1Department of Biological Sciences, Purdue University, 2Department of Oncology, University of Alberta, 3Cross Cancer Institute

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

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Neuroscience

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures
Matthew Moore *1, Yifan Hu *1, Sarah Woo 1, Dylan O'Hearn 1, Alexandru D. Iordan 2,3, Sanda Dolcos 1, Florin Dolcos 1,2,3
1Psychology Department, University of Illinois Urbana-Champaign, 2Neuroscience Program, University of Illinois Urbana-Champaign, 3Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

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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

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars
Mahtab Hassanpourfard 1, Xiaohui Sun 2, Amin Valiei 1, Partha Mukherjee 3, Thomas Thundat 1, Yang Liu 2, Aloke Kumar 4
1Department of Chemical and Material Engineering, University of Alberta, 2Department of Civil and Environmental Engineering, University of Alberta, 3Department of Mechanical Engineering, Texas A&M University, 4Department of Mechanical Engineering, University of Alberta

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

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Medicine

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

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

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Medicine

Murine Ileocolic Bowel Resection with Primary Anastomosis
Troy Perry 1, Anna Borowiec 1, Bryan Dicken 1, Richard Fedorak 2, Karen Madsen 2
1Department of Surgery, University of Alberta, 2Department of Medicine, University of Alberta

Ileocolic resection is commonly performed in several human diseases; however, little is known regarding the impact of intestinal resection on surgical illnesses. This article provides instruction on executing the procedure in mice with high success, providing a means to study the effects of ileocolic resection in models of disease.

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Environment

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry
Anna R. Bramucci 1, Leen Labeeuw 1, Teaghan J. Mayers 1, Julie A. Saby 1, Rebecca J. Case 1
1Department of Biological Sciences, University of Alberta

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

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Engineering

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates
Robert F. Peters 1,2, Luis Gutierrez-Rivera 1,2, Steven K. Dew 1,2, Maria Stepanova 1,2
1Department of Electrical and Computer Engineering, University of Alberta, 2National Institute for Nanotechnology, National Research Council of Canada

We describe the fabrication and characterization of nano-biological systems interfacing nanostructured substrates with immobilized proteins and aptamers. The relevant experimental steps involving lithographic fabrication of nanostructured substrates, bio-functionalization, and surface-enhanced Raman spectroscopy (SERS) characterization, are reported. SERS detection of surface-immobilized proteins, and probing of protein-ligand and aptamer-ligand binding is demonstrated.

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Neuroscience

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders
Surya T. Madabattula 1, Joel C. Strautman 1, Andrew M. Bysice 1, Julia A. O’Sullivan 1, Alaura Androschuk 1, Cory Rosenfelt 1, Kacy Doucet 1, Guy Rouleau 2, Francois Bolduc 1
1Department of Pediatrics, University of Alberta, 2Montreal Neurological Institute and Hospital, McGill University

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

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Bioengineering

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration
Sébastien Meghezi 1, Dawit G. Seifu 1,2, Nina Bono 1,3, Larry Unsworth 2,4,5, Kibret Mequanint 6, Diego Mantovani 1,2
1Laboratory for Biomaterials and Bioengineering, Department Min-Met-Materials Eng & CHU de Québec Research Center, Canada Research Chair I for the Innovation in Surgery, Laval University, 2NSERC CREATE Program for Regenerative Medicine (NCPRM), Laval University, 3Department Electronics, Information and Bioengineering, Politecnico di Milano, 4Department of Chemical and Materials Engineering, University of Alberta, 5National Institute for Nanotechnology, National Research Council (Canada), 6Department of Chemical and Biochemical Engineering, University of Western Ontario

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

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Biology

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)
Hilmar Strickfaden 1, Zhi Zhong Xu 1, Michael J. Hendzel 1
1Department of Oncology, Faculty of Dentistry and Medicine, University of Alberta

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

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Bioengineering

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii
Swayamdipta Bhaduri 1, Nandini Debnath 1, Sushanta Mitra 2, Yang Liu 3, Aloke Kumar 1
1Department of Mechanical Engineering, University of Alberta, 2Department of Mechanical Engineering, York University, 3Department of Civil and Environmental Engineering, University of Alberta

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

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Developmental Biology

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR
Milena S. Blanes *1, Stephen C.M. Tsoi *1, Michael K. Dyck 1
1Department of Agricultural, Food and Nutritional Science, University of Alberta

This protocol describes an accurate, inexpensive, rapid and non-toxic method to determine the sex of Day 30 porcine embryo using PCR method after grinding an embryo into powder without phenol chloroform extraction and DNA column purification.

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Medicine

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs
Richard R. Uwiera 1, Rabban Mangat 1, Sandra Kelly 1, Trina C. Uwiera 2, Spencer D. Proctor 1
1Department of Agricultural, Food and Nutritional Science, University of Alberta, 2Department of Surgery, University of Alberta

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

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Chemistry

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions
Mohameedyaseen Syedbasha 1, Janina Linnik 1,2,3, Deanna Santer 4, Daire O'Shea 5, Khaled Barakat 4,6, Michael Joyce 4, Nina Khanna 7, D. Lorne Tyrrell 4, Michael Houghton 4, Adrian Egli 1,8
1Applied Microbiology Research, Department of Biomedicine, University of Basel, 2Department of Biosystems Science and Engineering, ETH Zurich, and Swiss Institute of Bioinformatics, 3Swiss Institute of Bioinformatics, 4Li Ka Shing Institute for Virology, University of Alberta, 5Regional Infectious Diseases Unit, University of Edinburgh, 6Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 7Infection Biology, Department of Biomedicine, University of Basel, 8Clinical Microbiology, University Hospital Basel

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

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Developmental Biology

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform
Reza Salehi *1, Stephen C.M. Tsoi *1, Marcos G. Colazo 2, Divakar J. Ambrose 1,2, Claude Robert 3, Michael K. Dyck 1
1Department of Agricultural, Food and Nutritional Science, University of Alberta, 2Livestock Research Branch, Alberta Agriculture and Forestry, 3Laboratory of Functional Genomics of Early Embryonic Development, Université Laval

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

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Medicine

Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.
Shereen M. Hamza 1, John E. Hall 2
1Department of Pediatrics, University of Alberta, 2Department of Physiology & Biophysics, University of Mississippi Medical Center, 3Mississippi Center for Obesity Research

Anesthetized mice exhibit non-physiological systemic blood pressure, which precludes meaningful assessment of autonomic tone given the intimate relationship between blood pressure and the autonomic nervous system. Thus, a novel method to simultaneously record renal sympathetic nerve activity and blood pressure with intravenous infusion in conscious mice is outlined.

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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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Environment

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils
Sylvie A. Quideau 1, Anne C.S. McIntosh 2, Charlotte E. Norris 1, Emily Lloret 3, Mathew J.B. Swallow 4, Kirsten Hannam 5
1Department of Renewable Resources, University of Alberta, 2Department of Science, Augustana Faculty, University of Alberta, 3Laboratoire Génie Civil et géo-Environnement, Université de Lille, 4Department of Earth and Environmental Sciences, Mount Royal University, 5Forest Ecology & Production, Great Lakes Forestry Centre, Natural Resources Canada

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

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Immunology and Infection

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay
Salome Murinello 1, Stacey K. Moreno 1, Matthew S. Macauley 2, Susumu Sakimoto 1, Peter D. Westenskow 1,3, Martin Friedlander 1
1Department of Cell and Molecular Biology, The Scripps Research Institute, 2Department of Chemical Physiology, The Scripps Research Institute, 3The Lowy Medical Research Institute

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

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Biology

A Web Tool for Generating High Quality Machine-readable Biological Pathways
Miguel Ramirez-Gaona 1, Ana Marcu 1, Allison Pon 1, Jason Grant 1, Anthony Wu 1, David S. Wishart 1,2
1Department of Computer Science, University of Alberta, 2Department of Biological Sciences, University of Alberta

PathWhiz is a comprehensive, online pathway drawing tool for generating biochemical and biological pathways. It uses publicly accessible databases and easily expandable palettes consisting of pre-drawn pathway components. This protocol describes how to easily build new pathways, replicate and edit existing pathways, and propagate previously drawn pathways to different organisms.

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JoVE Core

Assessment of Pulmonary Capillary Blood Volume, Membrane Diffusing Capacity, and Intrapulmonary Arteriovenous Anastomoses During Exercise
Vincent Tedjasaputra 1,2, Sean van Diepen 3, Sophie É Collins 1,4, Wade M. Michaelchuk 1,2, Michael K. Stickland 1,5
1Division of Pulmonary Medicine, University of Alberta, 2Faculty of Physical Education and Recreation, University of Alberta, 3Divisions of Critical Care and Cardiology, University of Alberta, 4Faculty of Rehabilitation Medicine, University of Alberta, 5G.F. MacDonald Centre for Lung Health

To assess the pulmonary diffusion and vasculature responses to exercise, we describe the multiple-inspired oxygen diffusion capacity technique to determine capillary blood volume and membrane diffusing capacity, as well as agitated saline contrast echocardiography to assess the recruitment of intrapulmonary arteriovenous anastomoses.

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Education

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers
Richard Wang 1, Harris Wang 1, Zhixiang Wang 1
1Department of Medical Genetics, Signal Transduction Research Group, Faculty of Medicine and Dentistry, University of Alberta

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

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Engineering

In Situ Visualization of the Phase Behavior of Oil Samples Under Refinery Process Conditions
Cedric Laborde-Boutet 1, William C. McCaffrey 1
1Department of Chemical and Materials Engineering, University of Alberta

This article describes a setup and method for the in situ visualization of oil samples under a variety of temperature and pressure conditions that aim to emulate refining and upgrading processes. It is primarily used for studying isotropic and anisotropic media involved in the fouling behavior of petroleum feeds.

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Behavior

Neurodevelopmental Reflex Testing in Neonatal Rat Pups
Antoinette T. Nguyen 1, Edward A. Armstrong 1, Jerome Y. Yager 1
1Department of Pediatrics, University of Alberta

Behavioral testing is the gold standard for determining outcomes following brain injury, and can identify the presence of developmental disabilities in infants and children. Neurodevelopmental reflexes are an early indicator of these abnormalities. A host of easily accomplished developmental reflex tests in the neonatal rodent were developed and described here.

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Neuroscience

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils
Sara Breid 1, Maria E. Bernis 1, Julius B. Tachu 1, Maria C. Garza 2, Holger Wille 2, Gültekin Tamgüney 1
1German Center for Neurodegenerative Diseases (DZNE), 2Centre for Prions and Protein Folding Diseases & Department of Biochemistry, University of Alberta

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

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Biology

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass
Hieng Chiong Tie 1, Bing Chen 1, Xiuping Sun 1, Li Cheng 2,3, Lei Lu 1
1School of Biological Sciences, Nanyang Technological University, 2Bioinformatics Institute, 3School of Computing, National University of Singapore

The precise localization of Golgi residents is essential for understanding the cellular functions of the Golgi. However, conventional optical microscopy is unable to resolve the sub-Golgi structure. Here we describe the protocol for a conventional microscopy based super-resolution method to quantitatively determine the sub-Golgi localization of a protein.

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Bioengineering

A Test Bed to Examine Helmet Fit and Retention and Biomechanical Measures of Head and Neck Injury in Simulated Impact
Henry Y. Yu 1, Brooklynn M. Knowles 1, Christopher R. Dennison 1
1Department of Mechanical Engineering, University of Alberta

Using an anthropometric head and neck, optical fiber-based fit force transducers, an array of head acceleration and neck force/moment transducers, and a dual high speed camera system, we present a test bed to study helmet retention and effects on biomechanical measures of head and neck injury secondary to head impact.

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Cancer Research

Evaluating In Vitro DNA Damage Using Comet Assay
Yanxin Lu 1,2, Yang Liu 1, Chunzhang Yang 1
1Neuro-Oncology Branch, Center for Cancer Research, National Cancer Institute, 2Basic Medical Science Department, Zunyi Medical College-Zhuhai Campus

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

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Bioengineering

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation
Kyle M. Koss 1,2, Matthew A. Churchward 1,2, Andrea F. Jeffery 1,3, Vivian K. Mushahwar 1,2,4, Anastasia L. Elias 3, Kathryn G. Todd 1,2,5
1Department of Psychiatry, University of Alberta, 2Alberta Innovates-Health Solutions Interdisciplinary Team in Smart Neural Prostheses (Project SMART), University of Alberta, 3Department of Chemical and Materials Engineering, University of Alberta, 4Division of Physical Medicine and Rehabilitation, University of Alberta, 5Centre for Neuroscience, University of Alberta

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

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Engineering

All-electronic Nanosecond-resolved Scanning Tunneling Microscopy: Facilitating the Investigation of Single Dopant Charge Dynamics
Mohammad Rashidi 1,2, Wyatt Vine 1, Jacob A.J. Burgess 3,4,5, Marco Taucer 1,2,6, Roshan Achal 1, Jason L. Pitters 2, Sebastian Loth 3,4, Robert A. Wolkow 1,2
1Department of Physics, University of Alberta, 2National Institute for Nanotechnology, National Research Council of Canada, Edmonton, 3Max Planck Institute for the Structure and Dynamics of Matter, 4Max Planck Institute for Solid State Research, 5Department of Physics and Astronomy, University of Manitoba, 6Joint Attosecond Science Laboratory, University of Ottawa

We demonstrate an all-electronic method to observe nanosecond-resolved charge dynamics of dopant atoms in silicon with a scanning tunneling microscope.

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Developmental Biology

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks
George Merz 1, Valerie Schwenk 1, Ruchit Shah 2, Carolyn Salafia 1,2, Phillip Necaise 2, Michael Joyce 1, Tom Villani 3, Michael Johnson 3, Nick Crider 3
1The Institute for Basic Research, The New York State Office for People with Developmental Disabilities, 2Placental Analytics LLC, 3Visikol Inc.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

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Environment

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

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

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Immunology and Infection

Antigenic Liposomes for Generation of Disease-specific Antibodies
Kyle J. Bednar *1, Lakeya Hardy *2,3, Johanna Smeekens *3,4, Dharmendra Raghuwanshi 5, Shiteng Duan 6, Mike D. Kulis 3,4, Matthew S. Macauley 5,7
1Janssen R&D, 2Department of Microbiology and Immunology, University of North Carolina, 3UNC Food Allergy Initiative, University of North Carolina, 4Department of Pediatrics, University of North Carolina, 5Department of Chemistry, University of Alberta, 6Department of Molecular Medicine, Scripps Research Institute, 7Department of Medical Microbiology and Immunology, University of Alberta

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

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Medicine

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism
Sanaz Hatami 1, Christopher W. White 1, Martin Ondrus 1, Xiao Qi 1, Max Buchko 1, Sayed Himmat 1, Lily Lin 2, Katie Cameron 3, David Nobes 4, Hyun-Joong Chung 5, Jayan Nagendran 1,6, Darren H. Freed 1,3,6,7
1Department of Surgery, Faculty of Medicine, University of Alberta, 2Department of Pediatrics, Faculty of Medicine, University of Alberta, 3Department of Biomedical Engineering, Faculty of Medicine, University of Alberta, 4Department of Mechanical Engineering, Faculty of Engineering, University of Alberta, 5Department of Chemical and Materials Engineering, Faculty of Engineering, University of Alberta, 6Canadian National Transplant Research Program, 7Department of Physiology, Faculty of Medicine, University of Alberta

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

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JoVE Core

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)
Graeden Winkelaar 1, Catharine Trieber 1, Jitendra Kumar 1, Michael Overduin 1
1Department of Biochemistry, University of Alberta

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

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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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Behavior

A Vibrotactile Feedback Device for Seated Balance Assessment and Training
Andrew D. Williams 1, Albert H. Vette 1,2,3
1Department of Biomedical Engineering, University of Alberta, 2Department of Mechanical Engineering, University of Alberta, 3Glenrose Rehabilitation Hospital, Alberta Health Services

A sitting platform has been developed and assembled that passively destabilizes sitting posture in humans. During the user's stabilizing task, an inertial measurement unit records the device's motion, and vibrating elements deliver performance-based feedback to the seat. The portable, versatile device may be used in rehabilitation, assessment, and training paradigms.

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Developmental Biology

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis
Kevin H. Yoon 1, Sonya A. Widen 1,2, Melissa M. Wilson 1, Jennifer C. Hocking 2,3,4,5, Andrew J. Waskiewicz 1,2,6
1Department of Biological Sciences, University of Alberta, 2Women & Children's Health Research Institute, University of Alberta, 3Division of Anatomy, Department of Surgery, University of Alberta, 4Department of Cell Biology, University of Alberta, 5Department of Medical Genetics, University of Alberta, 6Neuroscience and Mental Health Research Institute, University of Alberta

Here, we present a standardized series of protocols to observe the superior ocular sulcus, a recently-identified, evolutionarily-conserved structure in the vertebrate eye. Using zebrafish larvae, we demonstrate techniques necessary to identify factors that contribute to the formation and closure of the superior ocular sulcus.

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Medicine

Brachial Artery Catheterization in Swine
Richard R.E. Uwiera 1, Amirali Toossi 2,3, Dirk G. Everaert 3,4, Trina C. Uwiera 5, Vivian K. Mushahwar 2,3,6
1Department of Agricultural, Food and Nutritional Science, University of Alberta, 2Neuroscience and Mental Health Institute, University of Alberta, 3Sensory Motor Adaptive Rehabilitation Technology (SMART) Network, University of Alberta, 4Division of Physical Medicine and Rehabilitation, University of Alberta, 5Division of Pediatric Surgery, Department of Surgery, University of Alberta, 6Division of Physical Medicine and Rehabilitation, University of Alberta

The video describes in detail the catheterization of the distal brachial artery in swine. This procedure accurately measures arterial blood pressure and is a simple and fast method to collect samples for arterial blood gas measurements.

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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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JoVE Methods Collection Highlights: Protein-Protein Interactions
Alastair Barr 1, Michael Overduin 2
1School of Life Sciences, University of Westminster, 2Department of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta

JoVE Methods Collection Highlights: Protein-Protein Interactions

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Behavior

Impact of High-intensity Interval Exercise and Moderate-Intensity Continuous Exercise on the Cardiac Troponin T Level at an Early Stage of Training
Haifeng Zhang 1,2, Jinlei Nie 3, Zhaowei Kong 4, Xiangui Zhu 1, Yang Liu 1, Qingde Shi 3
1Physical Education College, Hebei Normal University, 2Hebei Provincial Key Lab of Measurement and Evaluation in Human Movement and Bio-Information, 3School of Health Sciences and Sports, Macao Polytechnic Institute, 4Faculty of Education, University of Macau

Here, we present protocols of high-intensity interval and moderate-intensity continuous exercise to observe the response of circulating cardiac troponin T (cTnT) concentration to acute exercise over 10 days. The information may assist with clinical interpretations of post-exercise cTnT elevation and guide the prescription of exercise.

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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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Chemistry

18F-Labeling of Radiotracers Functionalized with a Silicon Fluoride Acceptor (SiFA) for Positron Emission Tomography
David Connolly *1, Justin J. Bailey *1, Harun Ilhan 2,3, Peter Bartenstein 2,3, Carmen Wängler 4, Björn Wängler 5, Melinda Wuest 1, Frank Wuest 1, Ralf Schirrmacher 1
1Department of Oncology, University of Alberta, 2Department of Nuclear Medicine, University Hospital of Munich, 3ENETS Centre of Excellence, Interdisciplinary Center of Neuroendocrine Tumors of the GastroEnteroPancreatic System, LMU Munich, 4Biomedical Chemistry, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University, 5Molecular Imaging and Radiochemistry, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University

The synthesis of fluorine-18 (18F) labeled radiopharmaceuticals for positron emission tomography typically requires months of experience. When incorporated into a radiotracer, the silicon-fluoride acceptor (SiFA) motif enables a simple 18F-labeling protocol that is independent of costly equipment and preparatory training, while reducing precursor quantity needed and utilizing milder reaction conditions.

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Behavior

Fully Automated Leg Tracking in Freely Moving Insects using Feature Learning Leg Segmentation and Tracking (FLLIT)
Animesh Banerjee 1, Shuang Wu 2, Li Cheng 3, Sherry Shiying Aw 1
1Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, 2Bioinformatics Institute, Agency for Science, Technology and Research, 3Department of Electrical and Computer Engineering, University of Alberta

We describe detailed protocols for using FLLIT, a fully automated machine learning method for leg claw movement tracking in freely moving Drosophila melanogaster and other insects. These protocols can be used to quantitatively measure subtle walking gait movements in wild type flies, mutant flies and fly models of neurodegeneration.

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JoVE Core

Cryogenic Liquid Jets for High Repetition Rate Discovery Science
Chandra B. Curry *1,2, Christopher Schoenwaelder *1,3, Sebastian Goede 4, Jongjin B. Kim 1, Martin Rehwald 5,6, Franziska Treffert 1,7, Karl Zeil 5, Siegfried H. Glenzer 1, Maxence Gauthier 1
1SLAC National Accelerator Laboratory, 2University of Alberta, 3Friedrich-Alexander-Universität Erlangen-Nürnberg, 4European XFEL, 5Helmholtz-Zentrum Dresden-Rossendorf, 6Technische Universität Dresden, 7Technische Universität Darmstadt

This protocol presents the operation and principles of micron-scale cylindrical and planar cryogenic liquid jets. Until now, this system has been used as a high repetition rate target in laser-plasma experiments. Anticipated cross-disciplinary applications range from laboratory astrophysics to material science, and eventually next-generation particle accelerators.

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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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Medicine

Focal Laser Ablation of Prostate Cancer: An Office Procedure
Wayne G. Brisbane 1, Shyam Natarajan 1, Alan Priester 1, Ely R. Felker 1, Adam Kinnaird 2, Leonard S. Marks 1
1University of California Los Angeles, 2University of Alberta

This article presents and describes an outpatient treatment for prostate cancer using focal laser ablation. Laser catheter placement is guided by MRI-ultrasound fusion imaging in a fashion similar to prostate needle biopsy. Treatment is monitored in real-time with a thermal probe, placed adjacent to the laser fiber.

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Immunology and Infection

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells
Shammy Raj 1, Lei Lu 2, Larry D. Unsworth 1
1Department of Chemical and Materials Engineering, Donadeo Innovation Centre for Engineering, University of Alberta, 2School of Life Science and Engineering, Southwest Jiaotong University

Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors.

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Bioengineering

Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture
Leshern Karamchand 1, Ashley Wagner 1, Syed Benazir Alam 1, Marianna Kulka 1,2
1Nanotechnology Research Center, National Research Council Canada, 2Department of Medical Microbiology and Immunology, University of Alberta

This protocol highlights a method to rapidly assess the biocompatibility of a crystalline nanocellulose (CNC)/agarose composite hydrogel biomaterial ink with mouse bone marrow-derived mast cells in terms of cell viability and phenotypic expression of the cell surface receptors, Kit (CD117) and high-affinity IgE receptor (FcεRI).

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Medicine

A Protocol for Roux-en-Y Gastric Bypass in Rats using Linear Staplers
Jerry T. Dang 1, Valentin Mocanu 1, Breanna Fang 2, Michael Laffin 1, Shahzeer Karmali 1, Karen Madsen 3, Daniel W. Birch 1
1Department of Surgery, University of Alberta, 2Faculty of Medicine and Dentistry, University of Alberta, 3Department of Medicine, University of Alberta

Roux-en-Y gastric bypass (RYGB) is performed to treat obesity and diabetes. However, the mechanisms underlying RYGB's efficacy are not fully understood, and studies are limited by technical difficulty leading to high mortality in animal models. This article provides instructions on how to perform RYGB in rats with high success rates.

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Medicine

Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP
Keir A. Forgie 1,2, Nicholas Fialka 3, Mubashir Khan 4, Max Buchko 1,2, Sanaz Hatami 4, Sayed Himmat 4, Xiao Qi 4, Xiuhua Wang 4, Katie-Marie Buswell 5, Ryan Edgar 5, Daina Domahidi 5, Darren H. Freed 1,2,6,7, Jayan Nagendran 1,2,6,7
1Division of Cardiac Surgery, Department of Surgery, Faculty of Medicine, University of Alberta, 2Mazankowski Alberta Heart Institute, 3Faculty of Medicine and Dentistry, University of Alberta, 4Department of Surgery, Faculty of Medicine, University of Alberta, 5Ray Rajotte Surgical-Medical Research Institute (SMRI), University of Alberta, 6Alberta Transplant Institute, 7Canadian Donation and Transplantation Research Program

This protocol describes a juvenile porcine model of orthotopic left lung allotransplantation designed for use with ESLP research. Focus is made on anesthetic and surgical techniques, as well as critical steps and troubleshooting.

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Medicine

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism
Keir A. Forgie 1,2, Nicholas Fialka 3, Max Buchko 1,2, Sayed Himmat 4, Sanaz Hatami 4, Xiao Qi 4, Xiuhua Wang 4, Katie-Marie Buswell 5, Ryan Edgar 5, Darren H. Freed 1,2,6,7, Jayan Nagendran 1,2,6,7
1Division of Cardiac Surgery, Department of Surgery, Faculty of Medicine, University of Alberta, 2Mazankowski Alberta Heart Institute, 3Faculty of Medicine and Dentistry, University of Alberta, 4Department of Surgery, Faculty of Medicine, University of Alberta, 5Ray Rajotte Surgical-Medical Research Institute (SMRI), University of Alberta, 6Alberta Transplant Institute, 7Canadian Donation and Transplantation Research Program

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

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Medicine

Transmission Electron Microscopy: A Surgical Pathology Tool for Neuroblastoma
Consolato M. Sergi *1,2,3,4, Janice Patry *1, Harry Coenraad 1, Jeff McClintock 1, Rod Nicholls 1, Hans-Jörg Steiner 5, Gregor Mikuz 5
1Anatomic Pathology Division of EORLA, Children's Hospital of Eastern Ontario, University of Ottawa, 2Department of Laboratory Medicine and Pathology, University of Alberta, 3Department of Pediatrics, University of Alberta, Stollery Children's Hospital, 4Department of Orthopedics, Tianyou Hospital, Wuhan University of Science and Technology, 5Institute of Pathology, Medical University of Innsbruck

Pediatric small round blue cell tumors are an intriguing and challenging collection of neoplasms. Therefore, transmission electron microscopy (TEM) and professional knowledge of pediatric tumors can be extremely valuable in surgical pathology. Here, we present a protocol to perform TEM for diagnosing neuroblastoma, one of the most common solid tumors in childhood.

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Engineering

Time-Resolved Photoluminescence Spectroscopy of Semiconductor Nanocrystals and Other Fluorophores
Alkiviathes Meldrum 1
1Department of Physics, University of Alberta

This paper presents an experimental how-to on time-resolved photoluminescence. The hardware used in many single photon-counting setups will be described and a basic how-to will be presented. This is intended to help students and experimenters understand the key system parameters and how to correctly set them in time-resolved photoluminescence setups.

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Medicine

Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction
Elizabeth Hillier 1,2, Jason Covone 1, Matthias G. Friedrich 1,3
1Faculty of Medicine and Health Sciences, McGill University, 2Faulty of Medicine and Dentistry, University of Alberta, 3Departments of Medicine and Diagnostic Radiology, McGill University

The assessment of microvascular function by oxygenation-sensitive cardiac magnetic resonance imaging in combination with vasoactive breathing maneuvers is unique in its ability to assess rapid dynamic changes in myocardial oxygenation in vivo and, thus, may serve as a critically important diagnostic technique for coronary vascular function.

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Cancer Research

Measurement of the Compressibility of Cell and Nucleus Based on Acoustofluidic Microdevice
Qibin Fu 1, Yan Zhang 1, Tuchen Huang 1, Yang Liu 1
1Sino-French Institute of Nuclear Engineering and Technology, Sun Yat-sen University

Here a protocol is presented to build a fast and non-destructive system for measuring cell or nucleus compressibility based on acoustofluidic microdevice. Changes in mechanical properties of tumor cells after epithelial-mesenchymal transition or ionizing radiation were investigated, demonstrating the application prospect of this method in scientific research and clinical practice.

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Neuroscience

Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning
Aislinn D. Maguire 1, Jason R. Plemel 1,2,3, Bradley J. Kerr 1,4,5
1Neuroscience and Mental Health Institute, University of Alberta, 2Department of Medicine, Division of Neurology, University of Alberta, 3Department of Medical Microbiology and Immunology, University of Alberta, 4Department of Pharmacology, University of Alberta, 5Department of Anesthesiology and Pain Medicine, University of Alberta

This paper describes an immunopanning protocol for adult mouse dorsal root ganglia. By adhering antibodies to culture plates, we can negatively select and remove non-neuronal cells. We show that the cultures are enriched for neurons using this protocol, allowing for an in-depth study of neuronal responses to manipulation.

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Medicine

A Piglet Perinatal Asphyxia Model to Study Cardiac Injury and Hemodynamics after Cardiac Arrest, Resuscitation, and the Return of Spontaneous Circulation
Eydis Oddsdottir Stenersen 1,2, Annette Olsen 2, Maria Melheim 3, Rønnaug Solberg 3,4, Ingrid Dannevig 5, Georg Schmölzer 6,7, Po-Yin Cheung 6,7, Britt Nakstad 8,9, Ola Didrik Saugstad 10,11, Arild Rønnestad 1,2, Anne Lee Solevåg 2
1Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, 2Department of Neonatal Intensive Care, Division of Pediatric and Adolescent Medicine, Oslo University Hospital Rikshospitalet, 3Department of Pediatric Research, Division of Paediatric and Adolescent Medicine, Oslo University Hospital Rikshospitalet, 4Department of Pediatrics, Vestfold Hospital Trust, 5Department of Anesthesiology, Oslo University Hospital Rikshospitalet, 6Department of Pediatrics, Faculty of Medicine and Dentistry, University of Alberta, 7Centre for the Studies of Asphyxia and Resuscitation, Neonatal Research Unit, Royal Alexandra Hospital, 8Division of Paediatric and Adolescent Medicine, Institute of Clinical Medicine, University of Oslo, 9Department of Paediatrics and Adolescent Health, University of Botswana, 10Department of Pediatric Research, Oslo University Hospital, University of Oslo, 11Department of Pediatrics, Robert H Lurie Medical Research Center, Northwestern University Chicago

This piglet model involves surgical instrumentation, asphyxiation until the cardiac arrest, resuscitation, and post-resuscitation observation. The model allows for multiple sampling per animal, and by using continuous invasive arterial blood pressure, ECG, and non-invasive cardiac output monitoring, it provides knowledge about hemodynamics and cardiac pathophysiology in perinatal asphyxia and neonatal cardiopulmonary resuscitation.

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Engineering

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

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

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Biochemistry

ABCG5/G8 Crystallization in a Lipidic Bicelle Environment for X-Ray Crystallography
Sarah Wazir 1, Danny Farhat 1, Mahalashmi Srinivasan 1,2, Jyh-Yeuan Lee 1
1Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 2Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta

This protocol describes a setup for the crystallization of the sterol transporter ABCG5/G8. ABCG5/G8 is reconstituted into bicelles for hanging-drop crystallization. The protocol does not require specialized materials or substrates, making it accessible and easy to adapt in any laboratory for determining the protein structure through X-ray crystallography.

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Medicine

Regenerative Peripheral Nerve Interface: Surgical Protocol for a Randomized Controlled Trial in Postamputation Pain
Emily Pettersen 1,2,3,4, Paolo Sassu 5, Francesca Alice Pedrini 1,5, Hannes Granberg 1,2, Carina Reinholdt 2,6, Juan Manuel Breyer 7, Aidan Roche 8, Andrew Hart 9,10, Adil Ladak 11, Hollie A. Power 11, Michael Leung 12, Michael Lo 12, Ian Valerio 13, Kyle R. Eberlin 13, Jason Ko 14, Gregory A. Dumanian 14, Theodore A. Kung 15, Paul Cederna 15, Max Ortiz-Catalan 1,4,16,17
1Center for Bionics and Pain Research, 2Center for Advanced Reconstruction of Extremities, Sahlgrenska University Hospital, 3Department of Electrical Engineering, Chalmers University of Technology, 4Bionics Institute, 5IV Clinica Ortoplastica, IRCCS Istituto Ortopedico Rizzoli, 6Department of Hand Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, 7Department of Orthopedic Surgery, Hand Unit, Worker Hospital, 8College of Medicine and Veterinary Medicine, The Queen's Medical Research Institute, The University of Edinburgh, 9Canniesburn Plastic Surgery Unit, Glasgow Royal Infirmary, 10College of Medicine, Veterinary & Life Sciences, The University of Glasgow, 11Division of Plastic Surgery, Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, 12Plastics and Reconstructive Surgery, Dandenong Hospital, Monash Health, 13Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital & Harvard Medical School, 14Division of Plastic Surgery, Department of Surgery, Northwestern Feinberg School of Medicine, 15Section of Plastic Surgery, Department of Surgery, Michigan Medicine, 16Medical Bionics Department, University of Melbourne, 17Prometei Pain Rehabilitation Center

Here, we describe the surgical procedure to perform Regenerative Peripheral Nerve Interface (RPNI) surgery for treating postamputation neuropathic pain in the context of an international, randomized controlled trial (RCT) (ClinicalTrials.gov, NCT05009394). The RCT compares RPNI with two other surgical techniques, namely, Targeted Muscle Reinnervation (TMR) and neuroma excision combined with intra-muscular transposition.

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Medicine

Targeted Muscle Reinnervation: Surgical Protocol for a Randomized Controlled Trial in Postamputation Pain
Emily Pettersen 1,2,3,4, Paolo Sassu 5, Francesca Alice Pedrini 1,5, Hannes Granberg 1,2, Carina Reinholdt 2,6, Juan Manuel Breyer 7, Aidan Roche 8, Andrew Hart 9,10, Adil Ladak 11, Hollie A. Power 11, Michael Leung 12, Michael Lo 12, Ian Valerio 13, Kyle R. Eberlin 13, Theodore A. Kung 14, Paul Cederna 14, Jason M. Souza 15,16, Oskar Aszmann 17, Jason Ko 18, Gregory A. Dumanian 18, Max Ortiz-Catalan 1,4,19,20
1Center for Bionics and Pain Research, 2Center for Advanced Reconstruction of Extremities, Sahlgrenska University Hospital, 3Department of Electrical Engineering, Chalmers University of Technology, 4Bionics Institute, 5IV Clinica Ortoplastica, IRCCS Istituto Ortopedico Rizzoli, 6Department of Hand Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, 7Department of Orthopedic, Hand Unit, Worker Hospital, 8College of Medicine and Veterinary Medicine, The Queen's Medical Research Institute, The University of Edinburgh, 9Canniesburn Plastic Surgery Unit, Glasgow Royal Infirmary, 10College of Medicine, Veterinary & Life Sciences, The University of Glasgow, 11Division of Plastic Surgery, Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, 12Plastics and Reconstructive Surgery, Dandenong Hospital, Monash Health, 13Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital &, Harvard Medical School, 14Section of Plastic Surgery, Department of Surgery, Michigan Medicine, 15Department of Surgery, Uniformed Services University of the Health Sciences and Walter Reed National Military Medical Center, 16Department of Plastic and Reconstructive Surgery, Ohio State University, 17Clinical Laboratory for Bionic Extremity Reconstruction, Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University Vienna, 18Division of Plastic Surgery, Department of Surgery, Northwestern Feinberg School of Medicine, 19Medical Bionics Department, University of Melbourne, 20Prometei Pain Rehabilitation Center

The protocol outlines the surgical procedure for the treatment of postamputation pain using Targeted Muscle Reinnervation (TMR). TMR will be compared with two other surgical techniques, specifically Regenerative Peripheral Nerve Interface (RPNI) and neuroma excision, followed by immediate burying within muscle under the context of an international, randomized controlled trial.

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Environment

Measurements of Soil Water Potential and Conductivity based on a Simple Evaporation Experiment using a Hydraulic Property Analyzer
Alessia J. Marchesan 1, Kris Guenette 1, Lewis K. Fausak 2, Guillermo Hernandez Ramirez 1
1Renewable Resources, University of Alberta, 2Faculty of Land and Food Systems, University of British Columbia

This article features a simple evaporation experiment using a hydraulic property instrument for a soil sample. Through efficient means, measurements can be taken over a series of days to generate high-quality data.

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