New York University View Institution's Website 32 articles published in JoVE Cancer Research Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures Abhijith Kuttanamkuzhi1, Libi Anandi1,2, Vaishali Chakravarty1,3, Mayurika Lahiri1 1Biology Department, Indian Institute of Science Education and Research, 2Department of Biology, Center for Genomics and Systems Biology, New York University, 3Enveda Therapeutics India Pvt Ltd. The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail. Biology Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Yuki X. Chen*1,5,6, Kelly Veerasammy*1,2, Jun Yin*3, Tenzin Choetso1,4, Tiffany Zhong7, Muniyat A. Choudhury1,4,5, Cory Weng1,4,5, Ethan Xu8, Mayan A. Hein9,10, Rinat Abzalimov2, Ye He1 1Advanced Science Research Center, Neuroscience Initiative, the City University of New York, Graduate Center New York, 2Advanced Science Research Center, Structural Biology Initiative, the City University of New York, Graduate Center, 3National Institute of Neurological Disorders and Stroke, National Institutes of Health, 4The City College of New York, CUNY, 5Macaulay Honors College, CUNY, 6Graduate School of Public Health and Health Policy, The City University of New York, 7Princeton University, 8Ardrey Kell High School, 9The Borough of Manhattan Community College, CUNY, 10Gallatin School of Individualized Study, New York University The aim of this protocol is to provide detailed guidance on the proper sample preparation for lipid and metabolite analysis in small tissues, such as the Drosophila brain, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging. Biology Laser Microdissection for Species-Agnostic Single-Tissue Applications Alyssa Woronik1,2, Karin Kiontke1, Raya S. Jallad1, R. Antonio Herrera3, David H. A. Fitch1 1Center for Developmental Genetics, New York University, 2Sacred Heart University, 3Baylor School A protocol is described that uses laser microdissection to isolate individual nematode tissues for RNA-sequencing. The protocol does not require species-specific genetic toolkits, allowing gene expression profiles to be compared between different species at the level of single-tissue samples. Genetics Rapid Isolation of Wild Nematodes by Baermann Funnel Sophia C. Tintori*1, Solomon A. Sloat*1, Matthew V. Rockman1 1Department of Biology and Center for Genomics & Systems Biology, New York University This protocol outlines a method for efficiently extracting live nematodes from natural substrates in the field. Biology Identification of Alternative Splicing and Polyadenylation in RNA-seq Data Gunjan Dixit1, Ying Zheng1, Brian Parker2, Jiayu Wen1 1Department of Genome Sciences, The John Curtin School of Medical Research, The Australian National University, 2Department of Biology, New York University Alternative splicing (AS) and alternative polyadenylation (APA) expand the diversity of transcript isoforms and their products. Here, we describe bioinformatic protocols to analyze bulk RNA-seq and 3' end sequencing assays to detect and visualize AS and APA varying across experimental conditions. Biology High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression Federica M. O. Sartori1, Cassandra Buzby1, Yevgeniy Plavskin1, Mark L. Siegal1 1Center for Genomics and Systems Biology, Department of Biology, New York University Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth. Neuroscience Preparation of the Rat Vocal Fold for Neuromuscular Analyses Charles Lenell1,2, Adrianna C. Shembel1, Aaron M. Johnson1 1NYU Voice Center, Department of Otolaryngology-Head & Neck Surgery, New York University School of Medicine, 2Communicative Sciences and Disorders, New York University This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study. Neuroscience Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats Gábor Kozák1, Tamás Földi1, Antal Berényi1,2 1MTA-SZTE "Momentum" Oscillatory Neuronal Networks Research Group, Department of Physiology, University of Szeged, 2New York University Neuroscience Institute, New York University This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats. Medicine Minimally Invasive Transverse Aortic Constriction in Mice Aung Moe Zaw1, Connor M. Williams2, Helen K. W. Law3, Billy Kwok Chong Chow1 1School of Biological Sciences, The University of Hong Kong, 2College of Arts and Science, New York University, 3Department of Health Technology and Informatics, Faculty of Health and Social Sciences, The Hong Kong Polytechnic University Minimally invasive transverse aortic constriction (MTAC) conserves the essentials of regular transverse aortic constriction (TAC) while eliminating the use of a ventilator with tracheal intubation. It proves to be a highly desirable method for high-throughput studies on left ventricular overload, particularly in translational studies. Behavior Ultrasound Images of the Tongue: A Tutorial for Assessment and Remediation of Speech Sound Errors Jonathan L. Preston1,2, Tara McAllister Byun3, Suzanne E. Boyce2,4, Sarah Hamilton4, Mark Tiede2, Emily Phillips2, Ahmed Rivera-Campos4, Douglas H. Whalen2,5,6 1Department of Communication Sciences and Disorders, Syracuse University, 2Haskins Laboratories, 3Department of Communicative Sciences and Disorders, New York University, 4Department of Communication Sciences and Disorders, University of Cincinnati, 5Program in Speech-Language-Hearing Sciences, City University of New York Graduate Center, 6Department of Linguistics, Yale University Ultrasound imaging can be used to display the shape and movements of the tongue in real time during speech. The images can be used to determine the nature of speech sound errors. Visual feedback of the tongue can be used to facilitate improvements in speech sound production in clinical populations. Behavior Using the Activity-based Anorexia Rodent Model to Study the Neurobiological Basis of Anorexia Nervosa Tara Gunkali Chowdhury1, Yi-Wen Chen1, Chiye Aoki1 1Center for Neural Science, New York University Here we present a protocol to induce activity-based anorexia (ABA) in female adolescent mice. ABA is a condition of hyperactivity evoked by imposing food restriction on rodents with access to a running wheel. This phenomenon is being used as a model to study the underlying neurobiology of anorexia nervosa. Medicine Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing Thomas Z. Thompson1, Farres Obeidin1, Alisa A. Davidoff2, Cody L. Hightower1, Christohper Z. Johnson1, Sonya L. Rice1, Rebecca-Lyn Sokolove1, Brandon K. Taylor1, John M. Tuck1, William G. Pearson, Jr.3,4 1Medical College of Georgia, Georgia Regents University, 2Department of Communicative Sciences and Disorders, New York University, 3Department of Cellular Biology & Anatomy, Georgia Regents University, 4Department of Otolaryngology, Georgia Regents University Coordinate mapping is a method of documenting salient features of hyolaryngeal biomechanics in the pharyngeal phase of swallowing. This methodology uses image analysis software to record coordinates of anatomical landmarks. These coordinates are imported into an excel macro and translated into kinematic variables of interest useful in dysphagia research. Behavior Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction C. R. Gallistel1, Fuat Balci1,2, David Freestone1,3, Aaron Kheifets1, Adam King4 1Department of Psychology, Rutgers University, 2Department of Psychology, Koç University, 3Center for Neural Science, New York University, 4Department of Mathematics & Computer Science, Fairfield University Fully automated system for measuring physiologically meaningful properties of the mechanisms mediating spatial localization, temporal localization, duration, rate and probability estimation, risk assessment, impulsivity, and the accuracy and precision of memory, in order to assess the effects of genetic and pharmacological manipulations on foundational mechanisms of cognition in mice. Biology The Use of Chemostats in Microbial Systems Biology Naomi Ziv1, Nathan J. Brandt1, David Gresham1 1Center for Genomics and Systems Biology, Department of Biology, New York University Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth. Immunology and Infection A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting Dumizulu L. Tembo1, Jacqui Montgomery1, Alister G. Craig2, Samuel C. Wassmer3 1Malawi-Liverpool-Wellcome Trust Clinical Research Programme, 2Liverpool School of Tropical Medicine, 3Department of Microbiology, Division of Medical Parasitology, New York University School of Medicine This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC. Biology Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization Julie Chaumeil1, Mariann Micsinai1,2,3,4, Jane A. Skok1 1Department of Pathology, New York University School of Medicine, 2New York University Center for Health Informatics and Bioinformatics, 3NYU Cancer Institute, 4Department of Pathology and Yale Cancer Center, Yale University School of Medicine Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH). Immunology and Infection Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice Wen-Chi Chen*1, Sung-Hyun Park*1, Carol Hoffman1, Cecil Philip1, Linda Robinson2, James West2, Gabriele Grunig1,3 1Department of Environmental Medicine, New York University School of Medicine, Tuxedo, 2Division of Allergy, Pulmonary, & Critical Care Medicine, Department of Medicine, Vanderbilt University Medical Center, 3Division of Pulmonary Medicine, New York University School of Medicine A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies. Neuroscience Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas Hui-Yi Hsiao*1, Robert J. Johnston Jr.*1, David Jukam*1, Daniel Vasiliauskas*1, Claude Desplan1, Jens Rister1 1Department of Biology, New York University The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation. Bioengineering Skin Tattooing As A Novel Approach For DNA Vaccine Delivery Yung-Nung Chiu1, Jared M. Sampson1, Xunqing Jiang1, Susan B. Zolla-Pazner2,3, Xiang-Peng Kong1 1Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 2Department of Pathology, New York University School of Medicine, 3Healthcare System, Veterans Affairs New York Harbor Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy. Neuroscience Measuring the Subjective Value of Risky and Ambiguous Options using Experimental Economics and Functional MRI Methods Ifat Levy1,2, Lior Rosenberg Belmaker1, Kirk Manson1, Agnieszka Tymula3, Paul W. Glimcher3,4,5 1Section of Comparative Medicine, Yale School of Medicine, 2Department of Neurobiology, Yale School of Medicine, 3Center for Neural Science, New York University, 4Department of Psychology, New York University, 5Department of Economics, New York University Using functional MRI and behavioral methods to determine the neural representation of the subjective value of risky and ambiguous options in the human brain. Neuroscience Extinction Training During the Reconsolidation Window Prevents Recovery of Fear Daniela Schiller1, Candace M. Raio2, Elizabeth A. Phelps3 1Departments of Psychiatry and Neuroscience, and Friedman Brain Institute, Mt. Sinai School of Medicine, 2Department of Psychology, New York University, 3Department of Psychology and Center for Neural Science, New York University Conditioned fear can be diminished through an inhibitory process called extinction, but can resurface under conditions such as the passage of time or exposure to stress. Our protocol presents a novel way of preventing fear recovery by introducing extinction during the reconsolidation window (the re-storage phase of a reactivated memory). Neuroscience Isolation and Culture of Human Fungiform Taste Papillae Cells Hakan Ozdener1, Andrew I. Spielman2, Nancy E. Rawson3 1Monell Chemical Senses Center, 2New York University College of Dentistry, 3AFB International We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Immunology and Infection A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation Mariko Kobayashi1, Ju-Youn Kim1, Vladimir Camarena2, Pamela C. Roehm3, Moses V. Chao2,4,5,6,7, Angus C. Wilson1, Ian Mohr1 1Department of Microbiology, New York University School of Medicine, 2Molecular Neurobiology Program, Skirball Institute for Biomolecular Medicine, New York University School of Medicine, 3Department of Otolaryngology, New York University School of Medicine, 4Department of Cell Biology, New York University School of Medicine, 5Department of Physiology and Neuroscience, New York University School of Medicine, 6Department of Psychiatry, New York University School of Medicine, 7Center for Neural Science, New York University School of Medicine The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins. Immunology and Infection Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers Kathleen C. Prins*1,2, Gaia Vasiliver-Shamis*2,3, Michael Cammer1,2, David Depoil1,2, Michael L. Dustin2, Catarina E. Hioe1,4 1Department of Pathology, New York University Langone School of Medicine, 2Program in Molecular Pathogenesis, Marty and Helen Kimmel Center for Biology and Medicine and Skirball Institute for Biomolecular Medicine, 3Laboratory of Molecular Immunogenetics, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 4Veteran Affairs New York Harbor Healthcare System This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation. Biology Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin Qing-Yun Tian1, Yun-Peng Zhao1, Chuan-ju Liu1,2 1Department of Orthopaedic Surgery, NYU Hospital for Joint Diseases, 2Department of Cell Biology, New York University School of Medicine We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable. Neuroscience How to Create and Use Binocular Rivalry David Carmel1,2, Michael Arcaro3,4, Sabine Kastner3,4, Uri Hasson3,4 1Department of Psychology, New York University, 2Centre for Neural Science, New York University, 3Department of Psychology, Princeton University, 4Neuroscience Institute, Princeton University Binocular rivalry occurs when the eyes are presented with different images at the same location: one image dominates while the other is suppressed, and dominance alternates periodically. Rivalry is useful for investigating perceptual selection and visual awareness. Here we describe several easy methods for creating and using binocular rivalry stimuli. Immunology and Infection Retroviral Transduction of T-cell Receptors in Mouse T-cells Shi Zhong1, Karolina Malecek1,2, Arianne Perez-Garcia1, Michelle Krogsgaard1 1NYU Cancer institute, New York University School of Medicine, 2Program in Structural Biology, New York University School of Medicine We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction Medicine Technique to Collect Fungiform (Taste) Papillae from Human Tongue Andrew I. Spielman*1, M. Yanina Pepino2, Roy Feldman3,4,5, Joseph G. Brand*4,6 1Department of Basic Science and Craniofacial Biology, College of Dentistry, New York University, 2Department of Internal Medicine and Department of Psychiatry, School of Medicine, Washington University in St. Louis, 3Veterans Affairs Medical Center, 4School of Dental Medicine, Department of Biochemistry, University of Pennsylvania-School of Medicine, 5Monell Chemical Senses Center, 6Monell Chemical Senses Center Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue. Immunology and Infection Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding David Almond1, Timothy Cardozo1 1Department of Pharmacology, School of Medicine, New York University The crown region of different V3 loop sequences of the surface envelope glycoprotein (gp120) of HIV-1 can be structurally characterized in many cases by in silico folding of positions 10 to 22 of the loop using a state-of-the-art ab initio folding algorithm. Here we demonstrate the folding and evaluation of this region of the V3 loop from the R2 strain of HIV-1, a uniquely neutralization sensitive strain with puzzling functional properties. Neuroscience SDS-PAGE/Immunoblot Detection of Aβ Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval Rebecca F. Rosen1, Yasushi Tomidokoro2, Jorge A. Ghiso3, Lary C. Walker1,4 1Yerkes National Primate Research Center, Emory University, 2Department of Neurology, Institute of Clinical Medicine, Tsukuba University, 3Department of Pathology, New York University School of Medicine, 4Department of Neurology, Emory University We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the Aβ peptide. Using this protocol, we consistently detect monomeric and multimeric Aβ in cortical tissue from humans with Alzheimer's pathology. Biology Fluorescence Activated Cell Sorting of Plant Protoplasts Bastiaan O. R. Bargmann1, Kenneth D. Birnbaum1 1Center for Genomics and Systems Biology, Department of Biology, New York University A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting. Biology Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse Santosha Vardhana1, Michael Dustin1 1Helen and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular, New York University - NYU Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.