Columbia University View Institution's Website 63 articles published in JoVE Bioengineering Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells Rosa L. Vincent1, Fangda Li2, Edward R. Ballister1, Nicholas Arpaia2,3, Tal Danino1,3,4 1Department of Biomedical Engineering, Columbia University, 2Department of Microbiology & Immunology, Vagelos College of Physicians and Surgeons, Columbia University, 3Herbert Irving Comprehensive Cancer Center, Columbia University, 4Data Science Institute, Columbia University This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells. Biology Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells Ines Cherkaoui1, Qian Du2, Dieter Egli2, Shivani Misra1,3, Guy A. Rutter1,4,5 1Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Cell Biology and Functional Genomics, 2Departments of Pediatrics, Naomi Berrie Diabetes Center, Obstetrics and Gynecology, Columbia Stem Cell Initiative, Columbia University Irving Medical Center, Columbia University, 3Department of Diabetes, Imperial College Healthcare NHS Trust, 4Faculté de Médicine, Université de Montréal, 5Lee Kong Chian Imperial Medical School, Nanyang Technological University This article presents a protocol for directed differentiation and functional analysis of β-cell like cells. We describe optimal culture conditions and passages for human pluripotent stem cells before generating insulin-producing pancreatic cells. The six-stage differentiation progresses from definitive endoderm formation to functional β-cell like cells secreting insulin in response to glucose. Bioengineering An Ex Vivo Porcine Model for Hydrodynamic Testing of Experimental Aortic Valve Procedures and Novel Medical Devices V. Reed LaSala1, Halil Beqaj2, Mingze Sun1, Sabrina Castagnini3, Senay Ustunel1, Elizabeth Cordoves2, Kavya Rajesh2, Sophia Jackman2, David Kalfa1,4 1Department of Surgery, Columbia University Medical Center, 2College of Physicians and Surgeons, Columbia University, 3School of Medicine and Surgery, University of Bologna, 4Division of Cardiac, Thoracic, and Vascular Surgery, Section of Pediatric and Congenital Cardiac Surgery, New York-Presbyterian Morgan Stanley Children’s Hospital, Columbia University Medical Center We present a method for mounting a porcine aortic valve on a pulse duplicator to test its hydrodynamic properties. This method can be used to determine the change in hydrodynamics after the application of an experimental procedure or novel medical device prior to use in a large animal model. Neuroscience An Open-Source Virtual Reality System for the Measurement of Spatial Learning in Head-Restrained Mice Clay Lacefield1,2, Hongtao Cai1, Huong Ho1, Carla Dias1, Hannah Chung1, René Hen1,2, Gergely F. Turi1,2 1Division of Systems Neuroscience, New York State Psychiatric Institute, 2Department of Psychiatry, Columbia University Here, we present a simplified open-source hardware and software setup for investigating mouse spatial learning using virtual reality (VR). This system displays a virtual linear track to a head-restrained mouse running on a wheel by utilizing a network of microcontrollers and a single-board computer running an easy-to-use Python graphical software package. Cancer Research Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids Samuel Flashner*1, Cecilia Martin*1,2, Norihiro Matsuura1, Masataka Shimonosono1, Yasuto Tomita1, Masaki Morimoto1, Ogoegbunam Okolo1, Victoria X. Yu1,3, Anuraag S. Parikh1,3, Andres J. P. Klein-Szanto4, Kelley Yan1,2, Joel T. Gabre1,5, Chao Lu1,6, Fatemeh Momen-Heravi1,7, Anil K. Rustgi1,5, Hiroshi Nakagawa1,2,5 1Herbert Irving Comprehensive Cancer Center, Columbia University, 2Organoid and Cell Culture Core, Columbia University Digestive and Liver Diseases Research Center, Columbia University, 3Department of Otolaryngology, Head and Neck Surgery, Columbia University, 4Histopathology Facility, Fox Chase Cancer Center, 5Division of Digestive and Liver Diseases, Department of Medicine, Columbia University, 6Department of Genetics and Development, Columbia University, 7Section of Oral, Diagnostic and Rehabilitation Sciences, College of Dental Medicine, Columbia University This protocol describes the key steps to generate and characterize murine oral-esophageal 3D organoids that represent normal, preneoplastic, and squamous cell carcinoma lesions induced via chemical carcinogenesis. Neuroscience Quantification of Immunostained Caspase-9 in Retinal Tissue Crystal K. Colón Ortiz1, Anna M. Potenski2, Kendra V. Johnson1, Claire W. Chen1, Scott J. Snipas3, Ying Y. Jean1, Maria I. Avrutsky*1, Carol M. Troy*1,4,5 1Department of Pathology & Cell Biology, Vagelos College of Physicians and Surgeons, Columbia University, 2Department of Molecular Pharmacology and Therapeutics, Vagelos College of Physicians and Surgeons, Columbia University, 3NCI-designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, 4Department of Neurology, Vagelos College of Physicians and Surgeons, Columbia University, 5The Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, Vagelos College of Physicians and Surgeons, Columbia University Presented here is a detailed immunohistochemistry protocol to identify, validate, and target functionally relevant caspases in complex tissues. Bioengineering A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation Lianna R. Gangi*1, Courtney A. Petersen*2, Sevan R. Oungoulian2, Eben G. Estell1, Krista M. Durney1, Jason T. Suh1, Gerard A. Ateshian1,2, Clark T. Hung1,3 1Department of Biomedical Engineering, Columbia University, 2Department of Mechanical Engineering, Columbia University, 3Department of Orthopedic Surgery, Columbia University The present protocol describes a friction testing device that applies simultaneous reciprocal sliding and normal load to two contacting biological counterfaces. Neuroscience In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility Claire W. Chen1, Anna M. Potenski2, Crystal K. Colón Ortiz1, Maria I. Avrutsky*1, Carol M. Troy*1,3,4 1Department of Pathology & Cell Biology; Vagelos College of Physicians and Surgeons, Columbia University, 2Department of Molecular Pharmacology and Therapeutics; Vagelos College of Physicians and Surgeons, Columbia University, 3Department of Neurology; Vagelos College of Physicians and Surgeons, Columbia University, 4The Taub Institute for Research on Alzheimer’s Disease and the Aging Brain; Vagelos College of Physicians and Surgeons, Columbia University Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO). Bioengineering Highly-Multiplexed Tissue Imaging with Raman Dyes Lixue Shi1, Mian Wei1, Wei Min1,2 1Department of Chemistry, Columbia University, 2Kavli Institute for Brain Science, Columbia University Electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes is a new platform for highly multiplexed epitope-based protein imaging. Here, we present a practical guide including antibody preparation, tissue sample staining, SRS microscope assembly, and epr-SRS tissue imaging. Bioengineering Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction Martin Liberman1, Miguel Chavez1, Trevor R. Nash1,2, Olaia F. Vila1,3, Gordana Vunjak-Novakovic1,2,4 1Department of Biomedical Engineering, Columbia University, 2Department of Medicine, Columbia University, 3Gladstone Institutes, 4College of Dental Medicine, Columbia University We describe a reproducible, automated, and unbiased imaging system for characterizing neuromuscular junction function using human engineered skeletal muscle tissue and optogenetic motoneurons. This system allows for the functional quantification of neuromuscular connectivity over time and detects diminished neuromuscular function caused by neurotoxins and myasthenia gravis patient serum. Bioengineering Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue Seyed Mohammad Mir1, Jiawen Chen1, Meghan R. Pinezich1, John D. O’Neill3, Brandon A. Guenthart4, Gordana Vunjak-Novakovic2, Jinho Kim1 1Department of Biomedical Engineering, Stevens Institute of Technology, 2Department of Biomedical Engineering, Columbia University, 3Department of Cell Biology, State University of New York Downstate Medical Center, 4Department of Cardiothoracic Surgery, Stanford University The protocol describes an imaging-enabled bioreactor that allows the selective removal of the endogenous epithelium from the rat trachea and homogenous distribution of exogenous cells on the lumen surface, followed by long-term in vitro culture of the cell-tissue construct. Bioengineering A Micropatterning Assay for Measuring Cell Chirality Haokang Zhang1,2, Kacey Ronaldson-Bouchard3, Gordana Vunjak-Novakovic3,4, Leo Q. Wan1,2,5,6 1Department of Biomedical Engineering, Rensselaer Polytechnic Institute, 2Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute, 3Department of Biomedical Engineering, Columbia University, 4Department of Medicine, Columbia University, 5Department of Biological Sciences, Rensselaer Polytechnic Institute, 6Center for Modeling, Simulation, and Imaging in Medicine, Rensselaer Polytechnic Institute We present a protocol for determining multicellular chirality in vitro, using the micropatterning technique. This assay allows for automatic quantification of the left-right biases of various types of cells and can be used for screening purposes. Neuroscience Standardized Data Acquisition for Neuromelanin-Sensitive Magnetic Resonance Imaging of the Substantia Nigra Garrett Salzman1, Jocelyn Kim1, Guillermo Horga*1,2, Kenneth Wengler*1,2 1New York State Psychiatric Institute, 2Department of Psychiatry, Columbia University This protocol shows how to acquire neuromelanin-sensitive magnetic resonance imaging data of the substantia nigra. Neuroscience Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability Crystal Colón Ortiz*1, Anna Potenski*2, Jaqueline M. Lawson1, Jade Smart1, Carol M. Troy1,3,4 1Department of Pathology & Cell Biology; Vagelos College of Physicians and Surgeons, Columbia University, 2Department of Molecular Pharmacology and Therapeutics; Vagelos College of Physicians and Surgeons, Columbia University, 3Department of Neurology; Vagelos College of Physicians and Surgeons, Columbia University, 4The Taub Institute for Research on Alzheimer’s Disease and the Aging Brain; Vagelos College of Physicians and Surgeons, Columbia University Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains. Biology Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles Nicole Comfort1, Kunheng Cai2, Tessa R. Bloomquist1, Madeleine D. Strait1, Anthony W. Ferrante Jr.2, Andrea A. Baccarelli1 1Department of Environmental Health Sciences, Columbia University Mailman School of Public Health, 2Department of Medicine, Naomi Berrie Diabetes Center, Columbia University We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma. Immunology and Infection Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes Veronica Jové*1, Krithika Venkataraman*1, Thomas M. Gabel2, Laura B. Duvall2 1Laboratory of Neurogenetics and Behavior, The Rockefeller University, 2Department of Biological Sciences, Columbia University The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested. Neuroscience Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity Alexander C. Whitebirch1 1Department of Neuroscience, Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity. Biochemistry A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers Dalton M. Pessôa Filho*1,2, Danilo A. Massini*2, Leandro O. C. Siqueira*2, Luiz Gustavo A. Santos*2, Camila M. T. Vasconcelos*2, Tiago A. F. Almeida*1,3, Mário A. C. Espada*4,5, Joana F. Reis*3,6, Francisco B. Alves*3, Fred J. DiMenna*7,8 1Department of Physical Education, São Paulo State University (UNESP) at Bauru, 2Institute of Bioscience, Graduate Program in Human Development and Technology, São Paulo State University (UNESP) at Rio Claro, 3Ciper, Faculdade de Motricidade Humana, Universidade de Lisboa, 4Department of Science and Technology, School of Education, Polytechnic Institute of Setúbal, 5Quality of Life Research Center, Polytechnic Institute of Santarem, 6Universidade Europeia at Lisbon, 7Division of Endocrinology, Diabetes and Bone Disease, Icahn School of Medicine at Mount Sinai, 8Department of Biobehavioral Sciences, Teachers College, Columbia University As opposed to measurement during free swimming, which presents inherent challenges and limitations, determination of important parameters of cardiorespiratory function for swimmers can be made using a more feasible and easier to administer tethered-swimming rapidly incremented protocol with gas exchange and ventilatory data collection. Bioengineering Biomechanical Testing of Murine Tendons Iden Kurtaliaj1,2, Mikhail Golman1,2, Adam C. Abraham1, Stavros Thomopoulos1,2 1Department of Orthopedic Surgery, Columbia University, 2Department of Biomedical Engineering, Columbia University The protocol describes efficient and reproducible tensile biomechanical testing methods for murine tendons through the use of custom-fit 3D printed fixtures. Chemistry High Resolution Physical Characterization of Single Metallic Nanoparticles Jessica Ettedgui1,2, Jacob Forstater1,2, Joseph W. Robertson1, John J. Kasianowicz1,3 1Physical Measurement Laboratory, National Institute of Standards and Technology, 2Department of Chemical Engineering, Columbia University, 3Department of Applied Physics and Applied Math, Columbia University Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules. Engineering Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins Neda M. Bassir Kazeruni*1, Stanislav Tsitkov*1, Henry Hess1 1Department of Biomedical Engineering, Columbia University We present a protocol to build molecular shuttles, where surface-adhered kinesin motor proteins propel dye-labelled microtubules. Weak interactions of the kinesins with the surface enables their reversible attachment to it. This creates a nanoscale system which exhibits dynamic assembly and disassembly of its components while retaining its functionality. Neuroscience A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum Regina T. Martuscello1, Elan D. Louis2,3,4, Phyllis L. Faust1 1Department of Pathology and Cell Biology, Columbia University, 2Division of Movement Disorders, Department of Neurology, Yale University, 3Department of Chronic Disease Epidemiology, Yale School of Public Health, Yale University, 4Center for Neuroepidemiology and Clinical Neurological Research, Yale School of Medicine, Yale University This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing. Developmental Biology Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle Wenyue Guan1, Lalanti Venkatasubramanian2, Myungin Baek3, Richard S. Mann2, Jonathan Enriquez1 1Institut de Génomique Fonctionnelle de Lyon, ENS de Lyon, CNRS, 2Departments of Biochemistry and Molecular Biophysics, and Neuroscience, Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University, 3Neuroscience Program, NYU School of Medicine Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps. Bioengineering An Additive Manufacturing Technique for the Facile and Rapid Fabrication of Hydrogel-based Micromachines with Magnetically Responsive Components Sau Yin Chin1,2, Yukkee Cheung Poh2, Anne-Céline Kohler2, Samuel K. Sia2 1Molecular Engineering Laboratory, Biomedical Sciences Institute, Agency for Science Technology and Research, 2Department of Biomedical Engineering, Columbia University An additive manufacturing strategy for processing UV-crosslinkable hydrogels has been developed. This strategy allows for the layer-by-layer assembly of microfabricated hydrogel structures as well as the assembly of independent components, yielding integrated devices containing moving components that are responsive to magnetic actuation. Immunology and Infection Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis William C. Cornell1, Chase J. Morgan1, Leslie Koyama1,2, Hassan Sakhtah1, Jennifer H. Mansfield2, Lars E.P. Dietrich1 1Department of Biological Sciences, Columbia University, 2Department of Biology, Barnard College, Columbia University We describe fixation, paraffin embedding, and thin sectioning techniques for microbial colony biofilms. In prepared samples, biofilm substructure and reporter expression patterns can be visualized by microscopy. Developmental Biology Dissection and Staining of Drosophila Pupal Ovaries Karen Sophia Park1, Dorothea Godt2, Daniel Kalderon1 1Department of Biological Sciences, Columbia University, 2Department of Cell and Systems Biology, University of Toronto The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries. Medicine Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia Mariya Zyablitskaya1, E. Laura Munteanu2, Takayuki Nagasaki1, David C. Paik1 1Department of Ophthalmology, Columbia University College of Physicians and Surgeons, 2Confocal and Specialized Microscopy Shared Resource, Herbert Irving Comprehensive Cancer Center, Columbia University This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using second harmonic generation imaging and differential scanning calorimetry. Neuroscience A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations Valerio Conti*1, Aurelie Carabalona*2,3,15, Emilie Pallesi-Pocachard2,3,4, Richard J. Leventer5,6,7, Fabienne Schaller2,3,8, Elena Parrini1, Agathe A. Deparis2,3, Françoise Watrin2,3, Emmanuelle Buhler2,3,8, Francesca Novara9, Stefano Lise10, Alistair T. Pagnamenta10, Usha Kini11, Jenny C. Taylor10, Orsetta Zuffardi9,12, Alfonso Represa2,3, David Antony Keays13, Renzo Guerrini1,14, Antonio Falace2,3, Carlos Cardoso2,3 1University of Florence, 2INSERM INMED, 3Aix-Marseille University, 4Plateforme Biologie Moléculaire et Cellulaire INMED, 5 Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo. Biochemistry Dissection of Human Retina and RPE-Choroid for Proteomic Analysis Thiago Cabral*1,2,7,8, Marcus A. Toral*3,4, Gabriel Velez3,4, James E. DiCarlo1,2, Anuradha M. Gore3, MaryAnn Mahajan3, Stephen H. Tsang1,2, Alexander G. Bassuk5,6, Vinit B. Mahajan3,9 1Barbara & Donald Jonas Stem Cell Laboratory, and Bernard & Shirlee Brown Glaucoma Laboratory, Department of Pathology & Cell Biology, Institute of Human Nutrition, College of Physicians and Surgeons, Columbia University, 2Edward S. Harkness Eye Institute, New York-Presbyterian Hospital, 3Omics Laboratory, Byers Eye Institute, Department of Ophthalmology, Stanford University, 4Medical Scientist Training Program, University of Iowa, 5Department of Pediatrics, University of Iowa, 6Department of Neurology, University of Iowa, 7Department of Ophthalmology, Federal University of Sao Paulo (UNIFESP), 8Department of Ophthalmology, Federal University of EspÍrito Santo (UFES), 9Palo Alto Veterans Administration, Palo Alto, CA The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis. Biochemistry Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms Danlin Yang1, Ajit Singh2, Helen Wu1, Rachel Kroe-Barrett1 1Department of Biotherapeutics Discovery, Immune Modulation and Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals, Inc., 2The Fu Foundation School of Engineering and Applied Science, Columbia University We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms. Bioengineering Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior Kai Wang1, Kam W. Leong2, Yong Yang1 1Department of Chemical and Biomedical Engineering, West Virginia University, 2Department of Biomedical Engineering, Columbia University A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented. Medicine Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases Stephen T. Armenti1, Jonathan P. Greenberg2, R. Theodore Smith1 1Department of Ophthalmology, NYU School of Medicine, 2Harkness Eye Institute, Columbia University The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy. 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. Bioengineering Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU) Julien Grondin1, Thomas Payen1, Shutao Wang1, Elisa E. Konofagou1,2 1Department of Biomedical Engineering, Columbia University, 2Department of Radiology, Columbia University This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region. Bioengineering Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect Daniel S. Oh1, Alia Koch1, Sidney Eisig1, Sahng Gyoon Kim2, Yoon Hyuk Kim3, Do-Gyoon Kim4, Jae Hyuck Shim5 1Oral and Maxillofacial Surgery, Columbia University, 2Endodontics, Columbia University, 3Mechanical Engineering, Kyung Hee University, South Korea, 4Orthodontics, The Ohio State University, 5Pathology, Weill Cornell Medical College A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels. Neuroscience Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization Jimena Baleriola1, Ying Jean1, Carol Troy1, Ulrich Hengst1 1College of Physicians and Surgeons, Columbia University RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains. Medicine Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models Mandy Stubbendorff*1,2, Xiaoqin Hua*1,2, Tobias Deuse1,2,3, Ziad Ali4,5, Hermann Reichenspurner2,3, Lars Maegdefessel6, Robert C. Robbins7, Sonja Schrepfer1,2,3,4 1Transplant and Stem Cell Immunobiology Lab, Cardiovascular Research Center, University Hospital Hamburg, 2Cardiovascular Research Center (CVRC) and DZHK University Hamburg, 3Department of Cardiovascular Surgery, University Heart Center Hamburg, 4Center for Interventional Vascular Therapy, Division of Cardiology, Columbia University, 5Cardiovascular Research Foundation, New York, 6Karolinska Institute, Stockholm, 7Department of Cardiothoracic Surgery, Stanford University School of Medicine, Falk Cardiovascular Research Center This video shows two models of intimal plaque development in murine arteries and emphasizes the differences in myointimal hyperplasia and atherosclerosis. Neuroscience A Single-fly Assay for Foraging Behavior in Drosophila Orel A. Zaninovich1, Susy M. Kim1, Cory R. Root1,2, David S. Green1,3, Kang I. Ko1,4, Jing W. Wang1 1Neurobiology Section, Division of Biological Sciences, University of California-San Diego, 2Department of Neuroscience, Columbia University, 3Dart NeuroScience, 4School of Dental Medicine, University of Pennsylvania In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. Bioengineering Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells Jason D. Vevea*1, Dana M. Alessi Wolken*1, Theresa C. Swayne2, Adam B. White2, Liza A. Pon1 1Department of Pathology and Cell Biology, Columbia University, 2Herbert Irving Comprehensive Cancer Center, Columbia University We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Biology Analyzing and Building Nucleic Acid Structures with 3DNA Andrew V. Colasanti1, Xiang-Jun Lu2, Wilma K. Olson1 1Department of Chemistry & Chemical Biology and BioMaPS Institute for Quantitative Biology, Rutgers - The State University of New Jersey, 2Department of Biological Sciences, Columbia University The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Neuroscience Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord Perrine Inquimbert1, Martin Moll2, Tatsuro Kohno3, Joachim Scholz2 1Département Nociception et Douleur, Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique (CNRS), 2Departments of Anesthesiology and Pharmacology, Columbia University, 3Department of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system. Medicine Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye Katherine J. Wert1,2, Jessica M. Skeie3,4, Richard J. Davis1, Stephen H. Tsang1,3, Vinit B. Mahajan3,4 1Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, Columbia University, 2Institute of Human Nutrition, College of Physicians & Surgeons, Columbia University, 3Omics Laboratory, University of Iowa, 4Department of Ophthalmology and Visual Sciences, University of Iowa This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye. Immunology and Infection Production and Titering of Recombinant Adeno-associated Viral Vectors Christina McClure*1, Katy L. H. Cole*1, Peer Wulff1, Matthias Klugmann2, Andrew J. Murray1,3 1School of Medical Sciences, College of Life Sciences and Medicine, University of Aberdeen, 2Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, University of New South Wales, 3Department of Biochemistry and Molecular Biophysics, Columbia University Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced. Medicine Mouse Eye Enucleation for Remote High-throughput Phenotyping Vinit B. Mahajan1,2, Jessica M. Skeie1,2, Amir H. Assefnia2,3, MaryAnn Mahajan1,2, Stephen H. Tsang2,4 1Department of Ophthalmology and Visual Sciences, University of Iowa, 2Omics Laboratory, University of Iowa, 3School of Dentistry, UCLA, 4Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens. Medicine Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice Jacqueline J. Glascock1, Erkan Y. Osman1, Tristan H. Coady2, Ferrill F. Rose1, Monir Shababi3, Christian L. Lorson3 1Department of Molecular Microbiology and Immunology, Bond Life Sciences Center, University of Missouri, 2Department of Biological Sciences, Columbia University, 3Department of Veterinary Pathobiology, Bond Life Sciences Center, University of Missouri This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice. Biology Evisceration of Mouse Vitreous and Retina for Proteomic Analyses Jessica M. Skeie1,2, Stephen H. Tsang3, Vinit B. Mahajan1,2 1Omics Laboratory, University of Iowa, 2Ophthalmology and Visual Sciences, University of Iowa, 3Harkness Eye Institute, Columbia University College of Physicians and Surgeons The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays. Immunology and Infection Recombinant Retroviral Production and Infection of B Cells Celia Keim1, Veronika Grinstein1, Uttiya Basu1,2 1Department of Microbiology and Immunology, Columbia University College of Physicians and Surgeons, 2Herbert Irving Comprehensive Cancer Center, Columbia University An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination. Biology Cargo Loading onto Kinesin Powered Molecular Shuttles Yolaine Jeune-Smith1, Ashutosh Agarwal2, Henry Hess2 1Department of Materials Science and Engineering, University of Florida, 2Department of Biomedical Engineering, Columbia University Molecular shuttles consisting of functionalized microtubules gliding on surface-adhered kinesin motor proteins can serve as a nanoscale transport system. Here, the assembly of a typical shuttle system is described. Neuroscience Basics of Multivariate Analysis in Neuroimaging Data Christian Georg Habeck1 1Department of Neurology, Columbia University The current article describes the basics of multivariate analysis and contrasts it to the more commonly used voxel-wise univariate analysis. Both types of analysis are applied to a clinical-neuroscience data set. Supplementary split-half simulations show better replication of the multivariate results in independent data sets. Neuroscience Preparation of Oligomeric β-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices Mauro Fa1, Ian J. Orozco1, Yitshak I. Francis1, Faisal Saeed1, Yimin Gong1, Ottavio Arancio1 1Taub Institute for Research on Alzheimer's Disease and Aging Brain, Columbia University One feature of Alzheimer's Disease is the elevation of Aβ1-42 peptide. Here we provide a protocol for preparing synthetic Aβ1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of Aβ-induced pathology and drug screening. Neuroscience The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow Zaman Mirzadeh1, Fiona Doetsch2,3, Kazunobu Sawamoto4, Hynek Wichterle2,5, Arturo Alvarez-Buylla1 1Department of Neurosurgery, The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco - UCSF, 2Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, 3Department of Neuroscience and Neurology, College of Physicians and Surgeons, Columbia University, 4Department of Developmental and Regenerative Biology, Nagoya City University Graduate School of Medical Sciences, 5Center for Motor Neuron Biology and Disease, College of Physicians and Surgeons, Columbia University The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Biology In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells Timothy J Petros1, Alexandra Rebsam1, Carol A Mason1,2 1Departments of Pathology and Cell Biology, and Neuroscience, Columbia University College of Physicians and Surgeons, 2Department of Ophthalmology, Columbia University College of Physicians and Surgeons Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties. Biology Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs Hui Zhang1,2, Niko G. Gubernator3,4, Minerva Yue1, Roland G. W. Staal1, Eugene V. Mosharov1, Daniela Pereira1, Vojtech Balsanek3, Paul A. Vadola3, Bipasha Mukherjee5, Robert H. Edwards5, David Sulzer1,2,6, Dalibor Sames3 1Departments of Neurology, Columbia University, 2Departments of Psychiatry and Pharmacology, Columbia University, 3Department of Chemistry, Columbia University, 4eMolecules, Inc., 5Departments of Neurology and Physiology, University of California School of Medicine, San Francisco, 6Division of Molecular Therapeutics, New York Psychiatric Institute A new means to measure neurotransmission optically using fluorescent dopamine analogs. Biology Primary Culture of Adult Rat Heart Myocytes Xianghua Xu1, Henry M. Colecraft1,2 1Department of Physiology and Cellular Biophysics, Columbia University, 2Department of Pharmacology, Columbia University In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements. Biology Drosophila Larval NMJ Immunohistochemistry Jonathan Brent1, Kristen Werner1, Brian D. McCabe1 1Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva. Biology Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae Wendy Imlach1, Brian D. McCabe1 1Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle. Biology Drosophila Larval NMJ Dissection Jonathan R. Brent1, Kristen M. Werner1, Brian D. McCabe1 1Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction. Biology Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG) Neela Zareen1, Lloyd A. Greene2 1Department of Biology, Columbia University, 2Department of Pathology and Cell Biology, Columbia University This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups. Biology Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons Hae Young Lee1, Lloyd A. Greene2, Carol A. Mason2,3, M. Chiara Manzini2,4 1Department of Genetics and Development, Columbia University, 2Department of Pathology and Cell Biology, Columbia University, 3Department of Neuroscience, Columbia University, 4Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse. Biology Microcontact Printing of Proteins for Cell Biology Keyue Shen1, Jie Qi1, Lance C. Kam1 1Department of Biomedical Engineering, Columbia University Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass. Biology Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring Vincent Ferrera1,2,3, Jack Grinband1,3, Tobias Teichert1, Franco Pestilli1, Stephen Dashnaw3, Joy Hirsch1,3 1Department of Neuroscience, Columbia University, 2Department of Psychiatry, Columbia University, 3Department of Radiology, Columbia University This presentation demonstrates the use of fMRI to study neural circuits that underlie decision-making. Simple perceptual tasks are combined with appetitive and aversive reinforcements to investigate how outcomes affect decision processes. Biology A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse Li Liu1, Karen Duff1 1Department of Pathology, Columbia University Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why Aβ or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse. Editorial Interview with Eric R. Kandel: From Memory, Free Will, and the Problem with Freud to Fortunate Decisions Eric R. Kandel, M.D.1,2 1Howard Hughes Medical Institute Research Laboratories, 2Center for Neurobiology and Behavior, Columbia University Eric R. Kandel shared the Nobel Prize for Physiology or Medicine with Arvid Carlsson and Paul Greengard in 2000 for their discoveries "concerning signal transduction in the nervous system." In this interview given at Hertie Foundation's Neuroforum 2008 on April 18, 2008 in Frankfurt, Germany, Nobel Prize Laureate Eric R. Kandel takes us on an enlighting journey ranging from memory, free will, "the problem with Freud", to scientific challenges and the rise of European science.