University of British Columbia View Institution's Website 67 articles published in JoVE Medicine Measuring Maxillary Posterior Tooth Movement: A Model Assessment using Palatal and Dental Superimposition Abdulraheem A. Alwafi1,2, Scott Panther2, Angela Lo2, Edwin H. Yen2, Bingshuang Zou2 1Department of Dental Public Health, Faculty of Dentistry, King Abdulaziz University, 2Department of Oral Health Science, Faculty of Dentistry, University of British Columbia This manuscript presents a comprehensive protocol to evaluate the three-dimensional (3D) movement of maxillary posterior teeth with clear aligners using digital model superimposition, an invaluable tool in orthodontics and dentofacial orthopedics. Developmental Biology An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios Kieran Maheden1, Karen Hwang1, Ipek Egilmez1, Nika Shakiba1 1School of Biomedical Engineering, University of British Columbia The present protocol describes a method for reverse poly-transfection of mouse embryonic stem cells during culture with 2i and LIF media. This method yields higher viability and efficiency than traditional forward transfection protocols, while also enabling one-pot optimization of plasmid ratios. Neuroscience Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia Morgan Towriss1, Jennifer Kim2, Annie Vogel Ciernia1 1Department of Biochemistry and Molecular Biology, Djavad Mowafaghian Centre for Brain Health, University of British Columbia, 2Graduate Program in Neuroscience, Djavad Mowafaghian Centre for Brain Health, University of British Columbia This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection. Developmental Biology Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters Jia Zhao*1, Shenghui Liang*1, Mitchell J. S. Braam1, Robert K. Baker1, Diepiriye G. Iworima1,2, Nina Quiskamp3, Timothy J. Kieffer1,2,4 1Department of Cellular & Physiological Sciences, Life Science Institute, University of British Columbia, 2School of Biomedical Engineering, University of British Columbia, 3STEMCELL Technologies Inc, 4Department of Surgery, University of British Columbia The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish. Chemistry Hydrogen Production and Utilization in a Membrane Reactor Alexandra R. Rousseau*1, Mia D. Stankovic*2, Curtis P. Berlinguette1,2,3,4 1Department of Chemical and Biological Engineering, The University of British Columbia, 2Department of Chemistry, The University of British Columbia, 3Stewart Blusson Quantum Matter Institute, The University of British Columbia, 4Canadian Institute for Advanced Research (CIFAR) Membrane reactors enable hydrogenation in ambient conditions without direct H2 input. We can track the hydrogen production and utilization in these systems using atmospheric mass spectrometry (atm-MS) and gas chromatography mass spectrometry (GC-MS). Bioengineering Rapid Development of Cell State Identification Circuits with Poly-Transfection Noreen Wauford1, Ross Jones1,2, Charles Van De Mark3, Ron Weiss1,3 1Department of Biological Engineering, Massachusetts Institute of Technology, 2School of Biological Engineering, University of British Columbia, 3Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology Complex genetic circuits are time-consuming to design, test, and optimize. To facilitate this process, mammalian cells are transfected in a way that allows the testing of multiple stoichiometries of circuit components in a single well. This protocol outlines the steps for experimental planning, transfection, and data analysis. Bioengineering Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells Nicolas Werschler1,2, Josef Penninger1,2,3,4 1School of Biomedical Engineering, University of British Columbia, 2Life Sciences Institute, University of British Columbia, 3Department of Medical Genetics, University of British Columbia, 4Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA) This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane. Neuroscience Assessing Changes in Synaptic Plasticity Using an Awake Closed-Head Injury Model of Mild Traumatic Brain Injury Brian R. Christie1,2, Allyson Gross1, Annika Willoughby1, Erin Grafe1, Justin Brand1, Emily Bosdachin1, Hannah M. O. Reid1, Crystal Acosta1, Eric Eyolfson1 1Division of Medical Sciences, University of Victoria, 2Island Medical Program, University of British Columbia Here, it is demonstrated how an awake closed-head injury model can be used for examining the effects of repeated mild traumatic brain injury (r-mTBI) on synaptic plasticity in the hippocampus. The model replicates important features of r-mTBI in patients and is used in conjunction with in vitro electrophysiology. Immunology and Infection Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging Katharine M. Ng1,2, Carolina Tropini1,2,3 1School of Biomedical Engineering, University of British Columbia, 2Department of Microbiology and Immunology, University of British Columbia, 3Humans and the Microbiome Program, Canadian Institute for Advanced Research (CIFAR) This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization. Chemistry Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry Lingxiao Chaihu1,2, Xiaopeng Yao1, Xiang Xu1,2, Zhongqin Zhu1, David D. Y. Chen3, Guanbo Wang1 1School of Chemistry and Materials Science, Nanjing Normal University, 2Institute for Cell Analysis, Shenzhen Bay Laboratory, 3Department of Chemistry, University of British Columbia Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation. Developmental Biology In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads Christina Rou Hsu1,2, Rain Xiong1,2, Kenji Sugioka1,2 1Department of Zoology, The University of British Columbia, 2Life Sciences Institute, The University of British Columbia Tissue complexities of multicellular systems confound the identification of causal relationship between extracellular cues and individual cellular behaviors. Here, we present a method to study the direct link between contact-dependent cues and division axes using C. elegans embryo blastomeres and adhesive polystyrene beads. Biochemistry PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays James W. Saville1, Luca A. Troman2, Franck Duong Van Hoa1 1Department of Biochemistry, University of British Columbia, 2School of Biochemistry, University of Bristol We present a method that combines membrane protein purification and reconstitution into peptidiscs in a single chromatographic step. Biotinylated scaffolds are used for direct surface attachment and measurement of protein-ligand interactions via biolayer interferometry. Immunology and Infection Chronic Salmonella Infection Induced Intestinal Fibrosis Bernard C. Lo1, Samuel B. Shin1, Melina Messing1, Kelly M. McNagny1 1The Biomedical Research Centre, University of British Columbia This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn’s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background. Immunology and Infection Probiotic Studies in Neonatal Mice Using Gavage Freddy Francis1, Natallia Varankovich2, Byron Brook1, Nelly Amenyogbe1, Rym Ben-Othman2, Bing Cai2, Danny Harbeson1, Aaron C. Liu1, Ben Dai2, Shelly McErlane4, Kris Andrews4, Tobias R. Kollmann*1,2, Pinaki Panigrahi*3 1Department of Experimental Medicine, University of British Columbia, 2Department of Pediatrics, University of British Columbia, 3Department of Epidemiology and Pediatrics, University of Nebraska Medical Centre, 4Animal Care Services, University of British Columbia This study details the process of gavaging precise amounts of probiotics to neonatal mice. The experimental set-up was optimized to include but is not limited to probiotic dosing, methods of administration, and quantification of bacteria in the intestines. Immunology and Infection A Controlled Mouse Model for Neonatal Polymicrobial Sepsis Byron Brook*1, Nelly Amenyogbe*1, Rym Ben-Othman2, Bing Cai2, Danny Harbeson1, Freddy Francis1, Aaron C. Liu1, Natallia Varankovich2, James Wynn3,4, Tobias R. Kollmann1,2 1Department of Experimental Medicine, University of British Columbia, 2Department of Pediatrics, Division of Infectious Diseases, University of British Columbia, 3Department of Pediatrics, College of Medicine, University of Florida, 4Department of Pathology, Immunology, and Laboratory Medicine, University of Florida This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice. Immunology and Infection Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy Jia C. Wang1, Madison Bolger-Munro1, Michael R. Gold1 1Department of Microbiology and Immunology, University of British Columbia We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation. Cancer Research Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay Michael M. Lizardo1,2, Poul H. Sorensen2,3 1Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2BC Cancer Agency, Provincial Health Services Authority, 3Department of Pathology and Laboratory Medicine, University of British Columbia The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope. Immunology and Infection Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System Ting-Yu Angela Liao1, Alice Lau1, Joseph Sunil1, Vesa Hytönen2, Zakaria Hmama1 1Division of Infectious Diseases, Department of Medicine and Vancouver Costal Health Research Institute, University of British Columbia, 2Institute of Biomedical Technology, University of Tampere A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches. Immunology and Infection Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation Lisa Kozicky1, Laura M. Sly1 1British Columbia Children's Hospital Research Institute, University of British Columbia Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages. Genetics Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis Alireza Lorzadeh1, Rodrigo Lopez Gutierrez1, Linda Jackson1, Michelle Moksa1, Martin Hirst1,2 1Department of Microbiology and Immunology, Michael Smith Laboratories Centre for High-Throughput Biology, University of British Columbia, 2Canada's Michael Smith Genome Science Center, BC Cancer Agency We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements. Neuroscience Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation Daniel Kai Long Tham1, Hakima Moukhles1 1Department of Cellular and Physiological Sciences, University of British Columbia Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report. Bioengineering Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel Corinne A. Hoesli1, Roger L. J. Kiang2, Kamini Raghuram2, René G. Pedroza3, Karen E. Markwick1, Antonio M. R. Colantuoni1, James M. Piret2 1Department of Chemical Engineering, McGill University, 2Michael Smith Laboratories & Department of Chemical and Biological Engineering, University of British Columbia, 3Michael Smith Laboratories & Department of Pharmaceutical Sciences, University of British Columbia This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications. Immunology and Infection System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis Xingji Zheng1, Yossef Av-Gay1 1Department of Medicine, University of British Columbia We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell. Neuroscience A Visual Guide to Sorting Electrophysiological Recordings Using 'SpikeSorter' Nicholas V. Swindale1, Catalin Mitelut1, Timothy H. Murphy2, Martin A. Spacek1,3 1Ophthalmology and Visual Sciences, University of British Columbia, 2Department of Psychiatry, University of British Columbia, 3Neurobiology, Biology II, LMU München The article shows how to use the program SpikeSorter to detect and sort spikes in extracellular recordings made with multi-electrode arrays. Engineering Experimental Procedure for Warm Spinning of Cast Aluminum Components Matthew J. Roy1, Daan M. Maijer2 1School of Mechanical, Aerospace, and Civil Engineering, University of Manchester, 2Materials Engineering, University of British Columbia An experimental protocol for instrumented warm rotary forming of cast aluminum alloys employing a bespoke industrially scaled apparatus is presented. Experimental considerations including thermal and mechanical effects are discussed, as well as similitude with full-scale processing of automotive wheels. Medicine Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device David Popok1, Christopher West1, Barbara Frias1, Andrei V. Krassioukov1,2 1International Collaboration on Repair Discoveries (ICORD), Faculty of Medicine, University of British Columbia, 2Department of Medicine, Division of Physical Medicine and Rehabilitation, University of British Columbia, GF Strong Rehabilitation Centre The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia. Immunology and Infection Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow Yifei Dong1, Arif A. Arif1, Grace F. T. Poon1, Blair Hardman1, Manisha Dosanjh1, Pauline Johnson1 1Microbiology and Immunology, University of British Columbia Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages. Biology Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging Gabriela Ziomek1, Cornelis van Breemen1, Mitra Esfandiarei1,2 1Department of Anaesthesiology, Pharmacology, and Therapeutics, University of British Columbia, 2Department of Biomedical Sciences, Midwestern University Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen. Biology Transfecting RAW264.7 Cells with a Luciferase Reporter Gene Sylvia T. Cheung1,2,3, Soroush Shakibakho1,2, Eva Y. So1,2, Alice L-F Mui1,2,3 1Immunity and Infection Research Centre, Vancouver Costal Health Research Institute, 2Department of Surgery, University of British Columbia, 3Department of Biochemistry and Molecular Biology, University of British Columbia Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell’s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells. Medicine Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration Mark J. Kearns1, Yingjin Wang3, John H. Boyd2 1Cardiovascular Surgery, UBC James Hogg Research Center, St. Paul's Hospital, 2Respiratory and Critical Care Medicine, UBC James Hogg Research Center, St. Paul's Hospital, 3UBC James Hogg Research Center, St. Paul's Hospital We describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The transplant configuration results in a partially loaded graft circulation, allowing direct functional assessment. This model may be employed for acute or chronic studies of function and immunologic status of the transplanted graft. Environment Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples Miguel I. Uyaguari-Diaz1, Jared R. Slobodan2, Matthew J. Nesbitt2, Matthew A. Croxen3, Judith Isaac-Renton1,3, Natalie A. Prystajecky1,3, Patrick Tang1,3 1Department of Pathology and Laboratory Medicine, Faculty of Medicine, The University of British Columbia, 2Coastal Genomics, 3British Columbia Public Health Microbiology and Reference Laboratory This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries. Engineering Visualization of High Speed Liquid Jet Impaction on a Moving Surface Yuchen Guo1, Sheldon Green1 1Department of Mechanical Engineering, University of British Columbia Two experimental devices for examining liquid jet impingement on a high-speed moving surface are described: an air cannon device and a spinning disk device. The apparatuses are used to determine optimal approaches to the application of liquid friction modifier (LFM) onto rail tracks for top-of-rail friction control. Medicine Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice Kyle Peake1, John Manning1, Coral-Ann Lewis1,2, Christine Barr1, Fabio Rossi2, Charles Krieger1,3 1Department of Biomedical Physiology and Kinesiology, Simon Fraser University, 2The Biomedical Research Centre, University of British Columbia, 3Division of Neurology, Department of Medicine, Neuromuscular Disease Unit, VHHSC We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system. Medicine Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay Christopher J. Johnson1, Christina M. Carlson2, Aaron R. Morawski3, Alyson Manthei4, Neil R. Cashman5 1USGS National Wildlife Health Center, 2Department of Soil Science, University of Wisconsin–Madison, 3Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 4Merial Veterinary Scholars Program, School of Veterinary Medicine, University of Wisconsin–Madison, 5Department of Neurology, University of British Columbia Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers. Behavior Operant Procedures for Assessing Behavioral Flexibility in Rats Anne Marie Brady1, Stan B. Floresco2 1 The ability to assess executive functions such as behavioral flexibility in rats is useful for investigating the neurobiology of cognition in both intact animals and disease models. Here we describe automated tasks for assessing strategy shifting and reversal learning, which are particularly sensitive to disruptions in prefrontal cortical networks. Medicine Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts Wolfgang Jäger1, Igor Moskalev1, Claudia Janssen1, Tetsutaro Hayashi1, Killian M. Gust1, Shannon Awrey1, Peter C. Black1 1Department of Urologic Sciences, University of British Columbia The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance. Immunology and Infection Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species Billie Velapatiño1, James E. A. Zlosnik1, Trevor J. Hird1, David P. Speert1 1Department of Pediatrics, Centre for Understanding and Preventing Infection in Children, University of British Columbia Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis. Neuroscience Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE) G. Stefano Brigidi1, Shernaz X Bamji1 1Department of Cellular and Physiological Sciences, Brain Research Centre, University of British Columbia The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues. Medicine Movement Retraining using Real-time Feedback of Performance Michael Anthony Hunt1 1Department of Physical Therapy, University of British Columbia Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time. Immunology and Infection Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols Rafi Chapanian1,2, Iren Constantinescu1,3, Donald E. Brooks1,2,4, Mark D. Scott1,2,3, Jayachandran Kizhakkedathu1,2,4 1Centre for Blood Research, University of British Columbia, 2Department of Pathology and Laboratory Medicine, University of British Columbia, 3Canadian Blood Services, University of British Columbia, 4Department of Chemistry, Life Sciences Centre, University of British Columbia The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards. Biology A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles Huan Bao1, Franck Duong1, Catherine S. Chan1 1Department of Biochemistry and Molecular Biology, University of British Columbia Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc. Immunology and Infection Depletion and Reconstitution of Macrophages in Mice Shelley B. Weisser1, Nico van Rooijen2, Laura M. Sly3 1Department of Graduate Studies, University of British Columbia, 2Department of Molecular Biology, Vrije Universiteit Amsterdam, 3Department of Pediatrics, University of British Columbia Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease. Medicine A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor Jae H.T. Lee1, Femke Streijger1, Seth Tigchelaar1, Michael Maloon1, Jie Liu1, Wolfram Tetzlaff1, Brian K. Kwon1,2 1International Collaboration on Repair Discoveries (ICORD), University of British Columbia, 2Department of Orthopaedics, University of British Columbia A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries. Biology Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry Helen Fleisig1, Judy Wong1 1Faculty of Pharmaceutical Sciences, University of British Columbia Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression. Neuroscience Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model Philip T.T. Ly*1, Fang Cai*1, Weihong Song1 1Department of Neuroscience, The University of British Columbia One of the pathological characteristics of AD is the formation of Amyloid β protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method. Neuroscience Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice Kelley Bromley-Brits*1, Yu Deng*1, Weihong Song1 1Department of Psychiatry, Brain Research Center, University of British Columbia The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models. Immunology and Infection Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut Frann Antignano*1, Sarah C. Mullaly*1, Kyle Burrows1, Colby Zaph1,2 1The Biomedical Research Centre, University of British Columbia, 2Department of Pathology and Laboratory Medicine, University of British Columbia Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response. Biology DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses Larissa A. Pikor*1,2, Katey S. S. Enfield*1,2, Heryet Cameron3, Wan L. Lam1,2,4 1Department of Integrative Oncology, BC Cancer Research Centre, 2Interdisciplinary Oncology Program, University of British Columbia - UBC, 3Photography/Video Production, Multi-Media Services, BC Cancer Agency, 4Department of Pathology and Laboratory Medicine, University of British Columbia - UBC This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis. Biology Purification of Progenitors from Skeletal Muscle Lin Yi1, Fabio Rossi1 1The Biomedical Research Centre, University of British Columbia Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle. Biology A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries Keith Mewis1, Marcus Taupp1, Steven J. Hallam1 1Microbiology and Immunology, University of British Columbia - UBC This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement. Immunology and Infection Facilitating the Analysis of Immunological Data with Visual Analytic Techniques David C. Shih1,2, Kevin C. Ho1, Kyle M. Melnick3, Ronald A. Rensink2,3, Tobias R. Kollmann1, Edgardo S. Fortuno III1 1Department of Paediatrics, Division of Infectious and Immunological Diseases, Child and Family Research Institute, University of British Columbia, 2Department of Computer Science, University of British Columbia, 3Department of Psychology, University of British Columbia Visual analytics (VA) is a new approach of analyzing data interactively. In this video, we discuss the data overload problem brought on by high-throughput biological experiments, and propose VA as a solution to such problem. The video demonstrates analysis within and between immunological datasets using a VA tool called Tableau. Biology Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris Maria Weidner1, Marcus Taupp1, Steven J. Hallam1 1Department of Microbiology and Immunology, University of British Columbia - UBC The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol. Biology Extraction of High Molecular Weight Genomic DNA from Soils and Sediments Sangwon Lee1, Steven J. Hallam1 1Department of Microbiology and Immunology, University of British Columbia - UBC A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described. Biology Large Insert Environmental Genomic Library Production Marcus Taupp1, Sangwon Lee1, Alyse Hawley1, Jinshu Yang1, Steven J. Hallam1 1Department of Microbiology and Immunology, University of British Columbia - UBC Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system. Biology DNA Extraction from 0.22 μM Sterivex Filters and Cesium Chloride Density Gradient Centrifugation Jody J. Wright1, Sangwon Lee1, Elena Zaikova1, David A. Walsh1, Steven J. Hallam1 1Department of Microbiology and Immunology, University of British Columbia - UBC We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 μm Sterivex filters, followed by cesium chloride density gradient centrifugation for purification. Biology Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc Patrick Cafferty1, Xiaojun Xie1, Kristen Browne1, Vanessa J. Auld1 1Department of Zoology, University of British Columbia - UBC Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells. Biology Small Volume (1-3L) Filtration of Coastal Seawater Samples David A. Walsh1, Elena Zaikova1, Steven J. Hallam1 1Department of Microbiology and Immunology, University of British Columbia - UBC This video documents small volume (~1 L) filtration of microbial biomass from the water column. Biology Large Volume (20L+) Filtration of Coastal Seawater Samples David A. Walsh1, Elena Zaikova1, Steven J. Hallam1 1Department of Microbiology and Immunology, University of British Columbia - UBC This video documents large volume (≥20 L) filtration of microbial biomass, ranging between 0.22μm and 2.7μm in diameter, from the water column. Biology Seawater Sampling and Collection Elena Zaikova1, Alyse Hawley1, David A. Walsh1, Steven J. Hallam1 1Department of Microbiology and Immunology, University of British Columbia - UBC This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses. Biology Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes Dee Brink1, Mary Gilbert1, Vanessa Auld1 1Department of Zoology, University of British Columbia - UBC We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans. Biology Identification of protein complexes with quantitative proteomics in S. cerevisiae Jesse Tzu-Cheng Chao1, Leonard J. Foster2, Christopher J. R. Loewen1 1Department of Cellular and Physiological Sciences, University of British Columbia - UBC, 2Department of Biochemistry and Molecular Biology, University of British Columbia - UBC Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p. Biology A high-throughput method to globally study the organelle morphology in S. cerevisiae Shabnam Tavassoli1, Jesse Tzu-Cheng Chao1, Christopher Loewen1 1Department of Cellular and Physiological Sciences, University of British Columbia - UBC GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast. Biology Microinjection of Xenopus Laevis Oocytes Sarah Cohen1, Shelly Au1, Nelly Panté1 1Department of Zoology, University of British Columbia - UBC Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy. Biology Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes Winco W.H. Wu1, Lindsay L. Weaver1, Nelly Panté1 1Department of Zoology, University of British Columbia - UBC The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs. Biology Methylated DNA Immunoprecipitation Kelsie L. Thu*1,2, Emily A. Vucic*1,3, Jennifer Y. Kennett1,3, Cameron Heryet5, Carolyn J. Brown5, Wan L. Lam1,2,3, Ian M. Wilson1,3 1Department of Cancer Genetics and Developmental Biology, BC Cancer Research Centre, 2Interdisciplinary Oncology Program, University of British Columbia - UBC, 3Department of Pathology and Laboratory Medicine, University of British Columbia - UBC, 4Photography/Video Production, Multi-Media Services, BC Cancer Agency, 5Department of Medical Genetics, Life Sciences Institute,, University of British Columbia - UBC This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC). Biology Single Cell Electroporation in vivo within the Intact Developing Brain D. Sesath Hewapathirane1,2, Kurt Haas1,2 1Brain Research Centre, University of British Columbia - UBC, 2Department of Cellular and Physiological Sciences, University of British Columbia - UBC Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole. Biology Use of Arabidopsis eceriferum Mutants to Explore Plant Cuticle Biosynthesis Lacey Samuels1, Allan DeBono1, Patricia Lam1, Miao Wen1, Reinhard Jetter1,2, Ljerka Kunst1 1Department of Botany, University of British Columbia - UBC, 2Department of Chemistry, University of British Columbia - UBC The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation but is also an important barrier against the entry of pathogenic microorganisms. In this video, we demonstrate the analysis of plant cuticle mutants identified by forward and reverse genetics approaches.
