University of North Carolina, Chapel Hill View Institution's Website 70 articles published in JoVE Medicine Extracellular Vesicle Tissue Factor Activity Assay Yohei Hisada1, Nigel Mackman1 1Division of Hematology, UNC Blood Research Center, University of North Carolina at Chapel Hill Here we describe an in-house extracellular vesicle tissue factor activity assay. Activity-based assays and antigen-based assays have been used to measure tissue factor in extracellular vesicles from human plasma samples. Activity-based assays have higher sensitivity and specificity than antigen-based assays. Medicine Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome Lauren C. Frazer1, Yukihiro Yamaguchi1, Corey M. Jania1, Wyatt E. Lanik2, Qingqing Gong3, Dhirendra K. Singh1, Stephen Mackay1, Natalia S. Akopyants1, Misty Good1 1Division of Neonatal-Perinatal Medicine, Department of Pediatrics, University of North Carolina at Chapel Hill, 2University of Nebraska College of Medicine, 3Department of Surgery, Washington University School of Medicine This protocol describes an in vitro model of necrotizing enterocolitis (NEC), which can be used for mechanistic studies into disease pathogenesis. It features a microfluidic chip seeded with intestinal enteroids derived from the human neonatal intestine, endothelial cells, and the intestinal microbiome of a neonate with severe NEC. Bioengineering Fabrication and Use of Dry Macroporous Alginate Scaffolds for Viral Transduction of T Cells Madelyn VanBlunk1, Pritha Agarwalla1,2, Sharda Pandit1,2, Yevgeny Brudno1,2,3 1Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, 2Comparative Medicine Institute, North Carolina State University, 3Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill Herein is a protocol for creating dry macroporous alginate scaffolds that mediate efficient viral gene transfer for use in genetic engineering of T cells, including T cells for CAR-T cell therapy. The scaffolds were shown to transduce activated primary T cells with >85% transduction. Immunology and Infection Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection Rajnikant Sharma1, Shekhar Yeshwante1, Quentin Vallé1, Maytham Hussein2, Varsha Thombare2, Sean Michael McCann1, Robert Maile3,4,5, Jian Li6, Tony Velkov2, Gauri Rao1 1UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, 2Department of Biochemistry & Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 3Department of Microbiology & Immunology, University of North Carolina School of Medicine, 4Department of Surgery, University of North Carolina at Chapel Hill, 5Curriculum in Toxicology and Environmental Medicine, University of North Carolina at Chapel Hill, 6Department of Microbiology, Monash Biomedicine Discovery Institute, Monash University A model mimicking the clinical scenario of burn injury and infection is necessary for furthering burn research. The present protocol demonstrates a simple and reproducible rat burn infection model comparable to that in humans. This facilitates the study of burn and infections following burn for developing new topical antibiotic treatments. Neuroscience Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy Felix A. Kyere*1,2, Ian Curtin*1,2, Oleh Krupa1,2, Carolyn M. McCormick1,2, Mustafa Dere3, Sarah Khan3,7, Minjeong Kim7, Tzu-Wen Winnie Wang4,5,6, Qiuhong He4,5,6, Guorong Wu3, Yen-Yu Ian Shih4,5,6, Jason L. Stein1,2 1UNC Neuroscience Center, University of North Carolina, Chapel Hill, 2Department of Genetics, University of North Carolina, Chapel Hill, 3Department of Psychiatry, University of North Carolina, Chapel Hill, 4Center for Animal Magnetic Resonance Imaging, The University of North Carolina at Chapel Hill, 5Biomedical Research Imaging Center, The University of North Carolina at Chapel Hill, 6Department of Neurology, The University of North Carolina at Chapel Hill, 7Department of Computer Science, The University of North Carolina at Greensboro This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph. Biochemistry Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions Brandy N. Curtis*1, Ellysa J. D. Vogt*2, Kevin S. Cannon1,3, Amy S. Gladfelter1,2,3 1Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, 2Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, 3Department of Biology, University of North Carolina at Chapel Hill Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports. Medicine Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery Sohini Ghosh1, David Chambers1, Adam R. Belanger1, Allen Cole Burks1, Christina MacRosty1, Anna Conterato1, Jason Long2, Benjamin Haithcock2, M. Patricia Rivera1, Jason A. Akulian1 1Section of Interventional Pulmonology, Division of Pulmonary and Critical Care Medicine, University of North Carolina at Chapel Hill, 2Section of Thoracic Surgery, Division of Cardiothoracic Surgery, University of North Carolina at Chapel Hill Presented here is a protocol for lung nodule localization using dye marking via electromagnetically navigated transthoracic needle access. The technique described here can be accomplished in the peri-operative period to optimize nodule localization and to successful resection when performing minimally invasive thoracic surgery. Neuroscience Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections Alex R. Eaker1, Katherine T. Baldwin1,2 1Neuroscience Center, University of North Carolina, Chapel Hill, 2Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill This protocol describes methods for sectioning, staining, and imaging free-floating tissue sections of the mouse brain, followed by a detailed description of the analysis of astrocyte territory volume and astrocyte territory overlap or tiling. Medicine Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models Jacquelyn J. Bower1,3, Zhenwei Song2, Liujiang Song1,2 1Department of Ophthalmology, University of North Carolina at Chapel Hill, 2Gene Therapy Center, University of North Carolina at Chapel Hill, 3Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes. Neuroscience Assessment of Sensory Thresholds in Dogs Using Mechanical and Hot Thermal Quantitative Sensory Testing Rachael M. Cunningham1,2, Rachel M. Park1,2, David Knazovicky3, B. Duncan X. Lascelles2,4,5,6, Margaret E. Gruen1,2,4 1Comparative Behavioral Research, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 2Translational Research in Pain (TRiP) Program, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 3Small Animal Orthopedic Surgery, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 4Comparative Pain Research and Education Center, College of Veterinary Medicine, North Carolina State University, 5Thurston Arthritis Center, UNC School of Medicine, University of North Carolina - Chapel Hill, 6Center for Translational Pain Research, Department of Anesthesiology, Duke University This work describes a standard protocol for mechanical and hot thermal quantitative sensory testing to evaluate the somatosensory system in dogs. Sensory thresholds are measured using an electronic von Frey anesthesiometer, pressure algometer, and hot contact thermode. Biology Automated Detection and Analysis of Exocytosis Fabio Urbina1, Stephanie L. Gupton1 1UNC Neuroscience Center, Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes. Biology Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging Stephanie Smith1, Alecia N. Septer1 1Department of Earth, Marine, and Environmental Sciences, University of North Carolina This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture. Medicine A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application Nicholas E. Buglak1,2,3,5, Edward S. M. Bahnson1,2,3,4,5 1Department of Surgery, Division of Vascular Surgery, University of North Carolina at Chapel Hill, 2Center for Nanotechnology in Drug Delivery, University of North Carolina at Chapel Hill, 3Curriculum in Toxicology & Environmental Medicine, University of North Carolina at Chapel Hill, 4Department of Cell Biology & Physiology, University of North Carolina at Chapel Hill, 5McAllister Heart Institute, University of North Carolina at Chapel Hill The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response. Developmental Biology Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging Russell H. Knutsen1, Leah M. Gober1, Joseph R. Sukinik1, Danielle R. Donahue2, Elise K. Kronquist1, Mark D. Levin1, Sean E. McLean3, Beth A. Kozel1 1Translational Vascular Medicine Branch, National Heart Lung and Blood Institute, National Institutes of Health, 2Mouse Imaging Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 3Division of Pediatric Surgery, Department of Surgery, University of North Carolina at Chapel Hill The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed. Environment RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points Shubham Rathore1, Jenni Hassert1, Courtney M. Clark-Hachtel2,3, Aaron Stahl1,4, Yoshinori Tomoyasu2, Elke K. Bushbeck1 1Department of Biological Sciences, University of Cincinnati, 2Department of Biology, Miami University, 3Department of Biology, University of North Carolina at Chapel Hill, 4Department of Neuroscience, The Scripps Research Institute RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior. Genetics Mapping Alzheimer's Disease Variants to Their Target Genes Using Computational Analysis of Chromatin Configuration Nana Matoba1,2, Ivana Y. Quiroga3, Douglas H. Phanstiel*3,4, Hyejung Won*1,2 1Department of Genetics, University of North Carolina, 2Neuroscience Center, University of North Carolina, 3Thurston Arthritis Research Center, University of North Carolina, 4Department of Cell Biology and Physiology, University of North Carolina We present a protocol to identify functional implications of non-coding variants identified by genome-wide association studies (GWAS) using three-dimensional chromatin interactions. Cancer Research Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports Kelly Pittman1, Shelton Earp1, Eric Ubil2 1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 2O'Neal Comprehensive Cancer Center, University of Alabama at Birmingham Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation. Biochemistry Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress Caitlin E Tolbert1, Lindsey Palmquist2, Hannah Lee Dixon2, Melissa C. Srougi2,3 1Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, 2Department of Chemistry, High Point University, 3Biotechnology Program and the Department of Molecular Biomedical Sciences, North Carolina State University This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways. Neuroscience Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes Blake A. Creighton*1, Theodore W. Ruffins*1, Damaris N. Lorenzo1 1Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics. Neuroscience Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells Luis J. Quintanilla1,2,3, Chia-Yu Yeh1,2, Hechen Bao1,2, Christina Catavero1,2,3, Juan Song1,2,3 1Department of Pharmacology, University of North Carolina Chapel Hill, 2Neuroscience Center, University of North Carolina Chapel Hill, 3Neuroscience Curriculum, University of North Carolina Chapel Hill The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit. Immunology and Infection Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates Lauren Speare1, Alecia N. Septer1 1Department of Marine Sciences, University of North Carolina, Chapel Hill Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population. Immunology and Infection Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System Susanna L. Harris1, Cesar A. Pelaez2, Elizabeth A. Shank1,2 1Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, 2Department of Biology, University of North Carolina at Chapel Hill Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments. Neuroscience Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips Smita R. Paranjape1,2, Tharkika Nagendran1,2, Valerie Poole3, Joseph Harris3, Anne Marion Taylor1,2,3 1UNC Neuroscience Center, 2UNC/NC State Joint Department of Biomedical Engineering, 3Xona Microfluidics, LLC This protocol demonstrates the use of compartmentalized microfluidic chips, injection molded in a cyclic olefin copolymer to cultured neurons differentiated from human stem cells. These chips are preassembled and easier-to-use than traditional compartmentalized poly(dimethylsiloxane) devices. Multiple common experimental paradigms are described here, including viral labeling, fluidic isolation, axotomy, and immunostaining. Developmental Biology Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo Trevor Henley1, Kandace Thomas1, Michael Bressan1 1Department of Cell Biology and Physiology, McAllister Heart Institute, University of North Carolina at Chapel Hill In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments. Biochemistry Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker Aleksandra Skrajna1, Xiao-Cui Yang1, Zbigniew Dominski1,2 1Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, 2Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes. Environment Production and Measurement of Organic Particulate Matter in a Flow Tube Reactor Yue Zhang1,2, Pengfei Liu1, Zhaoheng Gong1, Franz M. Geiger3, Scot T. Martin1,4 1School of Engineering and Applied Sciences, Harvard University, 2Department of Environmental Science and Engineering, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, 3Department of Chemistry, Northwestern University, 4Department of Earth and Planetary Sciences, Harvard University This paper describes the operation procedure for the flow tube reactor and related data collection. It shows the protocols for setting the experiments, recording data and generating the number-diameter distribution as well as the particle mass information, which gives useful information about chemical and physical properties of the organic aerosols. Environment Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber Yue Zhang1,2, Zhaoheng Gong1, Suzane de Sa1, Adam P. Bateman1, Yingjun Liu1, Yongjie Li1, Franz M. Geiger3, Scot T. Martin1,4 1School of Engineering and Applied Sciences, Harvard University, 2Department of Environmental Science and Engineering, Gillings School of Global Public Health, University of North Carolina, 3Department of Chemistry, Northwestern University, 4Department of Earth and Planetary Sciences, Harvard University This paper describes operation procedures for the Harvard Environmental Chamber (HEC) and related instrumentation for measuring gaseous and particle species. The environmental chamber is used to produce and study secondary organic species produced from the organic precursors, especially related to atmospheric organic particulate matter. Neuroscience Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons Tharkika Nagendran1,2, Valerie Poole3, Joseph Harris3, Anne Marion Taylor1,2,3 1UNC Neuroscience Center, 2UNC/NC State Joint Department of Biomedical Engineering, UNC, 3Xona Microfluidics, LLC This protocol describes the use of plastic chips to culture and compartmentalize primary murine neurons. These chips are preassembled, user-friendly, and compatible with high-resolution, live, and fluorescence imaging. This protocol describes how to plate rat hippocampal neurons within these chips and perform fluidic isolation, axotomy and immunostaining. Immunology and Infection Antigenic Liposomes for Generation of Disease-specific Antibodies Kyle J. Bednar*1, Lakeya Hardy*2,3, Johanna Smeekens*3,4, Dharmendra Raghuwanshi5, Shiteng Duan6, Mike D. Kulis3,4, Matthew S. Macauley5,7 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. Bioengineering Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers Anna M. Chiarella1, Tiffany A. Wang2, Kyle V. Butler3, Jian Jin3, Nathaniel A. Hathaway4 1Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, Curriculum in Genetics and Molecular Biology, University of North Carolina, 2College of Arts and Sciences, Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, University of North Carolina, 3Chemical Biology and Drug Discovery, Pharmacological Sciences and Oncological Sciences, Icahn School of Medicine at Mount Sinai, 4Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, Curriculum in Genetics and Molecular Biology, Lineberger Comprehensive Cancer Center, University of North Carolina Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner. Developmental Biology A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells Kimberly A. Peters1, Elizabeth Detmar1, Liz Sepulveda2, Corrina Del Valle2, Ruth Valsquier2, Anna Ritz2, Stephen L. Rogers1,3,4, Derek A. Applewhite2 1Department of Biology, The University of North Carolina at Chapel Hill, 2Department of Biology, Reed College, 3Carolina Center for Genome Sciences, The University of North Carolina at Chapel Hill, 4Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill Here we describe a contractility assay using Drosophila S2R+ cells. The application of an exogenous ligand, folded gastrulation (Fog), leads to the activation of the Fog signaling pathway and cellular contractility. This assay can be used to investigate the regulation of cellular contractility proteins in the Fog signaling pathway. Cancer Research Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer Milica Momcilovic1, Sean T. Bailey2, Jason T. Lee3, Charles Zamilpa3, Anthony Jones3, Gihad Abdelhady1, James Mansfield4, Kevin P. Francis5, David B. Shackelford1 1Division of Orthopaedic Surgery, University of California Los Angeles David Geffen School of Medicine In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology. Medicine Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds Kevin T. Sheets1, Juli R. Bagó1, Ivory L. Paulk1, Shawn D. Hingtgen1,2 1Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, 2Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking. Environment Adherence of Bacteria to Plant Surfaces Measured in the Laboratory Ann G. Matthysse1 1Department of Biology, University of North Carolina at Chapel Hill An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article. Engineering The Diffusion of Passive Tracers in Laminar Shear Flow Manuchehr Aminian1,2, Francesca Bernardi1, Roberto Camassa1, Daniel M. Harris1,3, Richard M. McLaughlin1 1Department of Mathematics, University of North Carolina at Chapel Hill, 2Department of Mathematics, Colorado State University, 3School of Engineering, Brown University A protocol for the study of the diffusion of passive tracers in laminar pressure-driven flow is presented. The procedure is applicable to various capillary pipe geometries. Biology Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining Wilber Escorcia1, Dana L. Ruter1,2,3, James Nhan1,2, Sean P. Curran1,2 1Leonard Davis School of Gerontology, University of Southern California, 2Molecular & Computational Biology Section, University of Southern California, 3Integrative Program for Biological & Genome Sciences, University of North Carolina at Chapel Hill Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues. Developmental Biology Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay Salma H. Azam1, Mitchell Smith2, Vivek Somasundaram2, Chad V. Pecot2,3 1Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, 2Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 3Department of Medicine, University of North Carolina at Chapel Hill This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis. Cancer Research Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors Elizabeth M. Corteselli1, James M. Samet2, Eugene A. Gibbs-Flournoy2,3 1Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, 2Environmental Public Health Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, 3Oak Ridge Institute for Science and Education This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress. Behavior Large Volume, Behaviorally-relevant Illumination for Optogenetics in Non-human Primates Leah C Acker1,2,3, Erica N. Pino1,4,5, Edward S. Boyden1,6,7, Robert Desimone1,7 1McGovern Institute, Massachusetts Institute of Technology, 2Harvard-MIT Division of Heath Sciences and Technology, 3Department of Anesthesiology, Duke University Medical Center, 4Department of Biology, Massachusetts Institute of Technology, 5Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, 6Media Lab and Department of Biological Engineering, Massachusetts Institute of Technology, 7Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology A protocol to build a tissue penetrating illuminator for delivering light over large volumes with minimal diameter is presented. Developmental Biology Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo Veronica Akle1, Nathalie Agudelo-Dueñas*1,2, Maria A. Molina-Rodriguez*1, Laurel Brianne Kartchner1,3,4,6, Annette Marie Ruth1,3,5,6, John M. González3, Manu Forero-Shelton2 1Laboratory of Neurosciences and Circadian Rhythms, School of Medicine, Universidad de los Andes, 2Biophysics Group, Department of Physics, Universidad de los Andes, 3Laboratory of Basic Medical Sciences, School of Medicine, Universidad de los Andes, 4Department of Microbiology and Immunology, University of North Carolina, 5Notre Dame Initiative for Global Development, University of Notre Dame, 6USAID Research and Innovation Fellowship program In this protocol, fluorescently labeled T. cruzi were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy. Cancer Research Sectioning Mammary Gland Whole Mounts for Lesion Identification Deirdre K Tucker1,2, Julie F Foley3, Schantel A Bouknight4, Suzanne E Fenton2 1Curriculum in Toxicology, University of North Carolina at Chapel Hill, 2National Toxicology Program Laboratory (NTPL), DNTP, National Institute of Environmental Health Sciences, 3Cellular and Molecular Pathology Branch, DNTP, National Institute of Environmental Health Sciences, 4Charles River Laboratories Inc. We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies. Biochemistry Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications Rachel A. Battaglia1, Parijat Kabiraj1, Helen H. Willcockson1, Melinda Lian1, Natasha T. Snider1 1Department of Cell Biology and Physiology, School of Medicine, University of North Carolina at Chapel Hill In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays. Biochemistry Measuring Protein Binding to F-actin by Co-sedimentation Jonathon A. Heier1, Daniel J. Dickinson2, Adam V. Kwiatkowski1 1Department of Cell Biology, University of Pittsburgh School of Medicine, 2Department of Biology, University of North Carolina at Chapel Hill This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction. Medicine Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals Phillip G. Durham1, Shumaila N. Hanif2, Lucia Garcia Contreras2, Ellen F. Young3, Miriam S. Braunstein3, Anthony J. Hickey1 1RTI International, 2Oklahoma University Health Science Center, 3University of North Carolina at Chapel Hill During development of drugs for pulmonary delivery, it is necessary to evaluate pharmacokinetics and efficacy in an animal model. We present a method to build a disposable aerosol dispersion system from of-the-shelf components that can be used to administer intrapulmonary dry powder aerosol to rodents. Biology Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins Kathryn A. Ramsey1,2, Zachary L. Rushton1, Camille Ehre1,3 1Marsico Lung Institute/CF Center, University of North Carolina at Chapel Hill, 2Telethon Kids Institute, University of Western Australia, 3Department of Pediatrics, University of North Carolina at Chapel Hill Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly. Developmental Biology Isolation and Characterization of Single Cells from Zebrafish Embryos Leigh Ann Samsa1,2, Nicole Fleming2,3, Scott Magness1, Li Qian2,3, Jiandong Liu2,3 1Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, 2McAllister Heart Institute, University of North Carolina at Chapel Hill, 3Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells. Chemistry A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions Marlon Lawrence1, Anze Testen1, Tilen Koklic2, Oliver Smithies1 1Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, 2Condensed Matter Physics Department, Laboratory of Biophysics, Jozef Stefan Institute We describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid (HAuCl4) with sodium thiocyanate (NaSCN). The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats. Behavior Measurement of Fronto-limbic Activity Using an Emotional Oddball Task in Children with Familial High Risk for Schizophrenia Sarah J. Hart1,2, Joseph J. Shaffer1,3, Joshua Bizzell1,2, Mariko Weber1,3, Mary A. McMahon2, Hongbin Gu1, Diana O. Perkins1, Aysenil Belger1,2 1Department of Psychiatry, University of North Carolina at Chapel Hill School of Medicine, 2Duke-UNC Brain Imaging and Analysis Center, Duke University Medical Center, 3Curriculum in Neurobiology, University of North Carolina at Chapel Hill This paper describes how to use the emotional oddball task and fMRI to measure brain activation in children and adolescents at familial high risk for schizophrenia (FHR). FMRI was used to measure differences in fronto-striato-limbic regions during an emotional oddball task. Children with FHR exhibited abnormal functional activation during adolescence. Medicine Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting Amy Weckle1,2, Allison E. Aiello3, Monica Uddin1,4, Sandro Galea5, Rebecca M. Coulborn6, Richelo Soliven7, Helen Meier6, Derek E. Wildman1,2 1Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 2Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 3Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina, 4Department of Psychology, University of Illinois at Urbana-Champaign, 5Department of Epidemiology, Mailman School of Public Health, Columbia University, 6Department of Epidemiology, University of Michigan School of Public Health, 7Center for Molecular Medicine and Genetics, Wayne State University School of Medicine We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting. Developmental Biology Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5 Li Wang1, Ziqing Liu1, Chaoying Yin1, Yang Zhou1, Jiandong Liu1, Li Qian1 1Department of Pathology and Laboratory Medicine, McAllister Heart Institute, University of North Carolina, Chapel Hill We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming. Medicine Isolation and Culture Expansion of Tumor-specific Endothelial Cells Lin Xiao1, James V. McCann1, Andrew C. Dudley1,2,3 1Department of Cell Biology & Physiology, University of North Carolina at Chapel Hill, 2Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 3McAllister Heart Institute, University of North Carolina at Chapel Hill We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models. Medicine Surgical Models of Gastroesophageal Reflux with Mice Jinxi He1,2, Yu Fang2,3, Xiaoxin Chen2,4 1Department of Thoracic Surgery, Ningxia Medical University General Hospital, 2Cancer Research Program, North Carolina Central University, 3Department of Thoracic & Cardiovascular Surgery, The First Affiliated Hospital of Chongqing Medical University, 4Department of Medicine, Center for Esophageal Disease and Swallowing, Division of Gastroenterology and Hepatology, University of North Carolina at Chapel Hill This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett’s esophagus and esophageal adenocarcinoma. Immunology and Infection Isolation and Transplantation of Different Aged Murine Thymic Grafts. Y. Maurice Morillon II*1, Fatima Manzoor*1, Bo Wang1, Roland Tisch1 1Department of Microbiology and Immunology, University of North Carolina at Chapel Hill This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo. Immunology and Infection Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice Erin C. Steinbach1,2, Gregory R. Gipson1, Shehzad Z. Sheikh1,3,4 1Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, 2Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, 3Department of Genetics, University of North Carolina at Chapel Hill, 4Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease. Developmental Biology Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes Adam C. Vandergriff1,2, Michael Taylor Hensley1,2, Ke Cheng1,2,3 1Department of Molecular Biomedical Sciences and Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University, 2Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, North Carolina State University, 3The Cyrus Tang Hematology Center, Soochow University The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time. Neuroscience Electroretinogram Analysis of the Visual Response in Zebrafish Larvae Jared D. Chrispell1, Tatiana I. Rebrik2, Ellen R. Weiss1 1Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, 2Department of Ophthalmology, Duke University We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo. Biology Protein Isolation from the Developing Embryonic Mouse Heart Valve Region Laura A. Dyer1, Yaxu Wu1, Cam Patterson2 1McAllister Heart Institute, University of North Carolina at Chapel Hill, 2New York-Presbyterian Hospital/Weill-Cornell Medical Center The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis. Neuroscience Deep Brain Stimulation with Simultaneous fMRI in Rodents John Robert Younce1,2,5, Daniel L Albaugh1,2,4, Yen-Yu Ian Shih1,2,3,4 1Department of Neurology, University of North Carolina, 2Biomedical Research Imaging Center, University of North Carolina, 3Department of Biomedical Engineering, University of North Carolina, 4Curriculum in Neurobiology, University of North Carolina, 5School of Medicine, University of North Carolina This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target. Biology Using Coculture to Detect Chemically Mediated Interspecies Interactions Elizabeth Anne Shank1 1Department of Biology, University of North Carolina at Chapel Hill Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media. Biology Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins Andrius Masedunskas1,2, Natalie Porat-Shliom1, Muhibullah Tora1, Oleg Milberg1,3, Roberto Weigert1 1Intracellular Membrane Trafficking Unit, Oral and Pharyngeal Cancer Branch National Institute of Dental and Craniofacial Research, National Institutes of Health, 2Department of Biology, University of North Carolina at Chapel Hill, 3Department of Chemical & Biochemical Engineering and Department of Biomedical Engineering, Rutgers University Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice. Biology Isolation of Embryonic Ventricular Endothelial Cells Laura A. Dyer1, Cam Patterson1 1McAllister Heart Institute, University of North Carolina at Chapel Hill Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis. Biology A Novel Ex vivo Culture Method for the Embryonic Mouse Heart Laura A. Dyer1, Cam Patterson1 1McAllister Heart Institute, University of North Carolina at Chapel Hill Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. Biology Microgavage of Zebrafish Larvae Jordan L. Cocchiaro1, John F. Rawls1 1Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill We present a novel method for microgavage of larval zebrafish utilizing standard embryo microinjection and stereomicroscopy equipment. We demonstrate that microgavage is a safe and efficient technique useful for delivering controlled amounts of diverse materials specifically into the larval zebrafish intestinal lumen. Biology Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates Faiza Waheed1,2, Pamela Speight1,2, Qinghong Dan1,2, Rafael Garcia-Mata3, Katalin Szaszi1,2 1Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital, 2Department of Surgery, University of Toronto, 3Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry. Medicine Eye Tracking Young Children with Autism Noah J. Sasson1, Jed T. Elison2 1School of Behavioral and Brain Sciences, University of Texas at Dallas, 2Carolina Institute for Developmental Disabilities, School of Medicine, University of North Carolina at Chapel Hill Eye tracking has long been used to study gaze patterns in typically-developing individuals, but recent technological advancements have made its use with clinical populations, including autism, more feasible. While eye-tracking young children with autism can offer insight into early symptom manifestations, it involves methodological challenges. Suggestions for best practices are provided. Immunology and Infection Generation of Organotypic Raft Cultures from Primary Human Keratinocytes Daniel Anacker1, Cary Moody1,2 1Department of Microbiology & Immunology, University of North Carolina-Chapel Hill, 2Lineberger Cancer Center, University of North Carolina-Chapel Hill An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue. Bioengineering Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique Shrirang Karve1,2, Michael E. Werner1,2, Natalie D. Cummings1,2, Rohit Sukumar1,2, Edina C. Wang1,2, Ying-Ao Zhang1,2, Andrew Z. Wang1,2 1Laboratory of Nano- and Translational Medicine, Department of Radiation Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, 2Carolina Center for Nanotechnology Excellence, University of North Carolina This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications. Biology Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c Adam J. Kole1, Elizabeth R.W. Knight2, Mohanish Deshmukh1 1Department of Cell and Developmental Biology, Neuroscience Center, University of North Carolina, 2Curriculum in Neurobiology, Neuroscience Center, University of North Carolina In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis. Medicine In Vivo Canine Muscle Function Assay Martin K. Childers1, Robert W. Grange2, Joe N. Kornegay3 1Department of Neurology and Wake Forest Institute for Regenerative Medicine, Wake Forest University, 2Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University, 3Departments of Pathology and Laboratory Medicine and Neurology and the Gene Therapy Center , University of North Carolina-Chapel Hill We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions. Biology Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays Pauline Chugh*1, Kristen Tamburro*1, Dirk P Dittmer1 1Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, Center for AIDS Research, University of North Carolina at Chapel Hill We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.
