Cornell University

58 articles published in JoVE

• Engineering

  
  Nanoscale Characterization of Liquid-Solid Interfaces by Coupling Cryo-Focused Ion Beam Milling with Scanning Electron Microscopy and Spectroscopy Taylor Moon¹, Michael Colletta¹, Lena F. Kourkoutis^{1,2 1}School of Applied and Engineering Physics, Cornell University, ²Kavli Institute at Cornell for Nanoscale Science Cryogenic Focused Ion Beam (FIB) and Scanning Electron Microscopy (SEM) techniques can provide key insights into the chemistry and morphology of intact solid-liquid interfaces. Methods for preparing high quality Energy Dispersive X-ray (EDX) spectroscopic maps of such interfaces are detailed, with a focus on energy storage devices.

• Biology

  
  Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes Mabel Taracena^1,2, Catherine Hunt¹, Pamela Pennington³, Deborah Andrew^4,5, Marcelo Jacobs-Lorena^5,6, Ellen Dotson¹, Michael Wells^{5,7,8 1}Division of Parasitic Diseases and Malaria, Entomology Branch, Centers for Disease Control and Prevention, ²Department of Entomology, Cornell University, ³Centro de Estudios en Biotecnologia, Universidad del Valle de Guatemala, ⁴Department of Cell Biology, Johns Hopkins School of Medicine, ⁵Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, ⁶Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health and Malaria Research Institute, ⁷Department of Cell Biology, Johns Hopkins School of Medicine, ⁸Biomedical Sciences Department, Idaho College of Osteopathic Medicine The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

• Neuroscience

  
  Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain Yusaku Hontani*¹, Najva Akbari*¹, Kristine E. Kolkman², Chunyan Wu^1,3, Fei Xia^1,4, Kibaek Choe¹, Grace Zhang Wang¹, Mariya Sokolova¹, Joseph R. Fetcho², Chris Xu^{1 1}School of Applied and Engineering Physics, Cornell University, ²Department of Neurobiology and Behavior, Cornell University, ³College of Veterinary Medicine, Cornell University, ⁴Meinig School of Biomedical Engineering, Cornell University Three-photon microscopy enables high-contrast fluorescence imaging deep in living biological tissues, such as mouse and zebrafish brains, with high spatiotemporal resolution.

• Environment

  
  Simulating Impacts of Ice Storms on Forest Ecosystems John L. Campbell¹, Lindsey E. Rustad¹, Charles T. Driscoll², Ian Halm³, Timothy J. Fahey⁴, Habibollah Fakhraei⁵, Peter M. Groffman^6,7, Gary J. Hawley⁸, Wendy Leuenberger⁹, Paul G. Schaberg^{10 1}Northern Research Station, U.S. Forest Service, Durham, NH, ²Department of Civil and Environmental Engineering, Syracuse University, ³Northern Research Station, U.S. Forest Service, North Woodstock, NH, ⁴Department of Natural Resources, Cornell University, ⁵Department of Civil and Environmental Engineering, Southern Illinois University, ⁶Advanced Science Research Center at the Graduate Center, City University of New York, ⁷Cary Institute of Ecosystem Studies, ⁸Rubenstein School of Environment and Natural Resources, University of Vermont, ⁹Department of Forestry and Natural Resources, University of Kentucky, ¹⁰Northern Research Station, U.S. Forest Service, Burlington, VT Ice storms are important weather events that are challenging to study because of difficulties in predicting their occurrence. Here, we describe a novel method for simulating ice storms that involves spraying water over a forest canopy during sub-freezing conditions.

• Biology

  
  LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging Hui Ji¹, Chun Han^{1 1}Weill Institute for Cell and Molecular Biology and Department of Molecular Biology and Genetics, Cornell University This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

• Biochemistry

  
  Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry Michael Vilkhovoy¹, David Dai¹, Sandra Vadhin*¹, Abhinav Adhikari*¹, Jeffrey D. Varner^{1 1}Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

• Developmental Biology

  
  Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure Brandon J. Gheller*¹, Jamie Blum*¹, Sharon Soueid-Baumgarten², Erica Bender¹, Benjamin D. Cosgrove², Anna Thalacker-Mercer^{1 1}Division of Nutritional Sciences, Cornell University, ²Meinig School of Biomedical Engineering, Cornell University We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

• Cancer Research

  
  Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells Hyeongsun Moon*¹, Leanne R. Donahue*¹, Dahihm Kim¹, Luye An¹, Andrew C. White^{1 1}Department of Biomedical Sciences, Cornell University The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

• Immunology and Infection

  
  Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting Jean K. Millet^1,2, Tiffany Tang³, Lakshmi Nathan³, Javier A. Jaimes⁴, Hung-Lun Hsu^3,5, Susan Daniel³, Gary R. Whittaker^{1 1}Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, ²INRA, Virologie et Immunologie Moléculaires, ³Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, ⁴Department of Microbiology, College of Agricultural and Life Sciences, Cornell University, ⁵Horae Gene Therapy Center, University of Massachusetts Medical School Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

• Biochemistry

  
  A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation Javier A. Jaimes*^1,2, Jean K. Millet*^1,3, Monty E. Goldstein^1,4, Gary R. Whittaker¹, Marco R. Straus^{1 1}Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, ²Department of Microbiology, College of Agricultural and Life Sciences, Cornell University, ³Virologie et Immunologie Moléculaires, Domaine de Vilvert, INRA, ⁴Department of Cell Biology and Molecular Genetics, University of Maryland We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

• Developmental Biology

  
  Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries Vera D. Rinaldi¹, Jordana C. Bloom², John C. Schimenti^{1,2 1}Department of Biomedical Sciences, Cornell University, ²Department of Molecular Biology and Genetics, Cornell University Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

• Genetics

  
  Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System Joseph C. Siefert^1,2, Emily A. Clowdus^1,2, Duane Goins¹, Amnon Koren³, Christopher L. Sansam^{1,2 1}Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, ²Department of Cell Biology, University of Oklahoma Health Sciences Center, ³Department of Molecular Biology and Genetics, Cornell University Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

• Developmental Biology

  
  Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea Divya A. Shiroor¹, Tisha E. Bohr¹, Carolyn E. Adler^{1 1}Department of Molecular Medicine, College of Veterinary Medicine, Cornell University The planarian Schmidtea mediterranea is an excellent model for studying stem cells and tissue regeneration. This publication describes a method to selectively remove one organ, the pharynx, by exposing animals to the chemical sodium azide. This protocol also outlines methods for monitoring pharynx regeneration.

• Biology

  
  A Murine Model of Vertical Sleeve Gastrectomy Darline Garibay¹, Bethany P. Cummings^{1 1}Department of Biomedical Sciences, Cornell University The following describes the performance of vertical sleeve gastrectomy in mice. This is a type of weight-loss surgery that involves removal of approximately 70% of the stomach.

• Chemistry

  
  Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor Sarah R. Nathan¹, Justin J. Wilson^{1 1}Department of Chemistry and Chemical Biology, Cornell University A protocol for the synthesis, purification, and characterization of a ruthenium-based inhibitor of mitochondrial calcium uptake is presented. A procedure to evaluate its efficacy in permeabilized mammalian cells is demonstrated.

• Immunology and Infection

  
  Identification of Plasmodesmal Localization Sequences in Proteins In Planta Cheng Yuan¹, Sondra G. Lazarowitz², Vitaly Citovsky^{1 1}Department of Biochemistry and Cell Biology, State University of New York, ²Department of Plant Pathology and Plant-Microbe Biology, Cornell University Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

• Engineering

  
  Measuring the Densities of Aqueous Glasses at Cryogenic Temperatures Chen Shen*¹, Ethan F. Julius*¹, Timothy J. Tyree*¹, Ritwik Dan¹, David W. Moreau², Robert Thorne^{2 1}Cornell University, ²Physics Department, Cornell University A protocol for determining the vitreous phase densities of micro- to pico-liter size drops of aqueous mixtures at cryogenic temperatures is described.

• Biochemistry

  
  Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development Robyn M. Johnston¹, Anne W. Sylvester², Michael J. Scanlon^{1 1}Plant Biology Section, School of Integrative Plant Science, Cornell University, ²Department of Developmental Genetics, University of Wyoming Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

• Medicine

  
  Transplantation Into the Mouse Ovarian Fat Pad Andrea Flesken-Nikitin¹, Blaine A. Harlan¹, Alexander Yu. Nikitin^{1 1}Department of Biomedical Sciences, Cornell University We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of Müllerian origin.

• Developmental Biology

  
  Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions Melinda L. Koyle¹, Madeline Veloz¹, Alec M. Judd¹, Adam C.-N. Wong^2,4, Peter D. Newell^2,5, Angela E. Douglas^2,3, John M. Chaston^{1,2 1}Department of Plant and Wildlife Sciences, Brigham Young University, ²Department of Entomology, Cornell University, ³Department of Molecular Biology and Genetics, Cornell University, ⁴Division of Infectious Diseases, Boston Children's Hospital, Harvard Medical School, ⁵Biological Sciences, SUNY Oswego A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

• Immunology and Infection

  
  The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids Daniel E. Rothschild¹, Tara Srinivasan², Linette A. Aponte-Santiago¹, Xiling Shen^2,3,4, Irving C. Allen^{1 1}Department of Biomedical Sciences and Pathobiology, Virginia Maryland College of Veterinary Medicine, Virginia Tech, ²Department of Biomedical Engineering, Cornell University, ³School of Electrical and Computer Engineering, Cornell University, ⁴Department of Biomedical Engineering, Duke University Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

• Biology

  
  High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots Thomas B. Jacobs¹, Gregory B. Martin^{1,2 1}Boyce Thompson Institute for Plant Research, ²Section of Plant Pathology & Plant-Microbe Biology, School of Integrative Plant Science, Cornell University Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

• Neuroscience

  
  Conditions Affecting Social Space in Drosophila melanogaster Alison R. McNeil¹, Sam N. Jolley¹, Adesanya A. Akinleye², Marat Nurilov², Zulekha Rouzyi², Austin J. Milunovich³, Moria C. Chambers³, Anne F. Simon^{1 1}Department of Biology, University of Western Ontario, ²Department of Biology, York College/CUNY, ³Department of Entomology, Cornell University The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

• Medicine

  
  Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells Sachi Horibata¹, Tommy V. Vo², Venkataraman Subramanian³, Paul R. Thompson³, Scott A. Coonrod^{1 1}Department of Baker Institute for Animal Health, Cornell University, ²Department of Molecular Biology and Genetics, Cornell University, ³Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

• Immunology and Infection

  
  Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster Sarah Khalil¹, Eliana Jacobson¹, Moria C. Chambers¹, Brian P. Lazzaro^{1 1}Department of Entomology, Cornell University Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

• Neuroscience

  
  In vivo Optogenetic Stimulation of the Rodent Central Nervous System Michelle M. Sidor¹, Thomas J. Davidson², Kay M. Tye³, Melissa R. Warden⁴, Karl Diesseroth^2,5, Colleen A. McClung^{1 1}Department of Psychiatry, University of Pittsburgh Medical Center, ²Department of Bioengineering, Stanford University, ³Department of Brain and Cognitive Sciences, Picower Institute for Learning and Memory, Massachusetts Institute of Technology, ⁴Department of Neurobiology and Behavior, Cornell University, ⁵Department of Psychiatry and Behavioral Sciences, Stanford University Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

• Neuroscience

  
  A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord Matthew J. Farrar^1,2, Chris B. Schaffer^{2 1}Department of Neurobiology and Behavior, Cornell University, ²Department of Biomedical Engineering, Cornell University In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

• Biology

  
  A Strategy for Sensitive, Large Scale Quantitative Metabolomics Xiaojing Liu¹, Zheng Ser¹, Ahmad A. Cluntun^1,2, Samantha J. Mentch^1,2, Jason W. Locasale^{1,2 1}Division of Nutritional Sciences, Cornell University, ²Field of Biochemistry, and Molecular Cell Biology, Cornell University Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

• Engineering

  
  Measuring Spatially- and Directionally-varying Light Scattering from Biological Material Todd Alan Harvey¹, Kimberly S. Bostwick^2,3, Steve Marschner^{4 1}Department of Biomedical Science, Cornell University, ²Department of Ecology and Evolutionary Biology, Cornell University, ³Cornell University Museum of Vertebrates, ⁴Department of Computer Science, Cornell University We present a non-destructive method for sampling spatial variation in the direction of light scattered from structurally complex materials. By keeping the material intact, we preserve gross-scale scattering behavior, while concurrently capturing fine-scale directional contributions with high-resolution imaging. Results are visualized in software at biologically-relevant positions and scales.

• Biology

  
  Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines Laura A. Martin¹, Marco Seandel^{1 1}Department of Surgery, Weill Cornell Medical College A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

• Medicine

  
  Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model Sharanya Iyengar¹, Yariv Houvras^2,3, Craig J. Ceol^{1 1}Program in Molecular Medicine and Department of Cancer Biology, University of Massachusetts Medical School, ²Departments of Surgery and Medicine, Weill Cornell Medical College, ³Departments of Surgery and Medicine, New York Presbyterian Hospital A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

• Biology

  
  Procedure for Fabricating Biofunctional Nanofibers Jereme Doss¹, Omotunde Olubi¹, Biswajit Sannigrahi¹, M. D. Williams², Deepti Gadi³, Barbara Baird³, Ishrat Khan^{1 1}Department of Chemistry, Clark Atlanta University, ²Department of Physics, Clark Atlanta University, ³Department of Chemistry and Chemical Biology, Cornell University An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.

• Bioengineering

  
  Continuously-stirred Anaerobic Digester to Convert Organic Wastes into Biogas: System Setup and Basic Operation Joseph G. Usack¹, Catherine M. Spirito¹, Largus T. Angenent^{1 1}Department of Biological and Environmental Engineering, Cornell University Laboratory-scale anaerobic digesters allow scientists to research new ways of optimizing existing applications of anaerobic biotechnology and to evaluate the methane producing potential of various organic wastes. This article introduces a generalized model for the construction, inoculation, operation, and monitoring of a laboratory-scale continuously stirred anaerobic digester.

• Bioengineering

  
  Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples Andrew D. Hughes¹, Jeff Mattison¹, John D. Powderly^2,3, Bryan T. Greene^2,3, Michael R. King^{1 1}Department of Biomedical Engineering, Cornell University, ²BioCytics, Inc., ³Carolina BioOncology Institute, PLLC Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

• Medicine

  
  The α-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva Cynthia L. Bristow¹, Mariya A. Babayeva¹, Rozbeh Modarresi¹, Carole P. McArthur², Santosh Kumar³, Charles Awasom⁴, Leo Ayuk⁴, Annette Njinda⁵, Paul Achu⁵, Ronald Winston^{6 1}Department of Medicine, Weill Cornell Medical College, ²Department of Oral Biology, University of Missouri-Kansas City-School of Dentistry, ³Department of Pharmacology and Toxicology, University of Missouri Kansas City- School of Pharmacy, ⁴Regional Hospital, Bamenda, NWP, Cameroon, ⁵Mezam Polyclinic HIV/AIDS Treatment Center, Cameroon, ⁶Institute for Human Genetics and Biochemistry A CD4 enumeration method, the α-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The α-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

• Bioengineering

  
  Silk Film Culture System for in vitro Analysis and Biomaterial Design Brian D. Lawrence¹, Zhi Pan¹, Michael D. Weber¹, David L. Kaplan², Mark I. Rosenblatt^{1 1}Margaret M. Dyson Vision Research Institute, Weill Cornell Medical College, ²Department of Biomedical Engineering, Tufts University Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

• Bioengineering

  
  A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke Long-Xi Yu¹, Boris G. Dzikovski², Jack H. Freed^{2 1}CDCF-AOX Lab, ²National Biomedical Center for Advanced ESR Technology (ACERT), Department of Chemistry and Chemical Biology, Cornell University Spin-trapping ESR spectroscopy was used to study the effect of plant antioxidants lycopene, pycnogenol and grape seed extract on scavenging gas-phase free radicals in cigarette smoke.

• Neuroscience

  
  Neural Tube Closure in Mouse Whole Embryo Culture Jason Gray¹, M. Elizabeth Ross^{1 1}Department of Neurology/Neuroscience, Weill Cornell Medical College A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

• Biology

  
  Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation Maria L. Lombardi¹, Monika Zwerger¹, Jan Lammerding^{1,2 1} We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

• Immunology and Infection

  
  Derivation of Thymic Lymphoma T-cell Lines from Atm^-/- and p53^-/- Mice Rasika Jinadasa¹, Gabriel Balmus¹, Lee Gerwitz¹, Jamie Roden¹, Robert Weiss¹, Gerald Duhamel^{1 1}Department of Biomedical Sciences, Cornell University In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

• Medicine

  
  Single Port Donor Nephrectomy David B Leeser*¹, James Wysock*², S Elena Gimenez², Sandip Kapur¹, Joseph Del Pizzo*^{2 1}Surgery, Weill Cornell Medical College of Cornell University, ²Urology, Weill Cornell Medical College of Cornell University Single port laparoscopic surgery is changing the standard of care in surgical care like nothing since the laparoscopic technique was introduced 20 years ago. We present out technique of single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in 100 patients.

• Biology

  
  Isolation of Valvular Endothelial Cells Russell A. Gould¹, Jonathan T. Butcher^{1 1}Department of Biomedical Engineering, Cornell University We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

• Neuroscience

  
  Combining Computer Game-Based Behavioural Experiments With High-Density EEG and Infrared Gaze Tracking Keith J. Yoder^1,2, Matthew K. Belmonte^{1,3 1}Department of Human Development, Cornell University, ²Social Sciences Division, University of Chicago, ³National Brain Research Centre, Manesar, India Procedures for recording high-density EEG and gaze data during computer game-based cognitive tasks are described. Using a video game to present cognitive tasks enhances ecological validity without sacrificing experimental control.

• Biology

  
  Isolation and Culture of Avian Embryonic Valvular Progenitor Cells Gretchen Mahler¹, Russell Gould¹, Johnathan Butcher^{1 1}Department of Biomedical Engineering, Cornell University This article will provide a method for isolating and culturing quail or chicken HH14^- valve endocardial cells and HH25 valve cushion mesenchymal cells.

• Biology

  
  An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications Huseyin C. Yalcin^1,2, Akshay Shekhar¹, Ajinkya A. Rane¹, Jonathan T. Butcher^{1 1}Department of Biomedical Engineering, Cornell University, ²Current Address: Mechanical Engineering Department, Dogus University In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

• Biology

  
  Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties Helgi I. Ingólfsson¹, R. Lea Sanford¹, Ruchi Kapoor¹, Olaf S. Andersen^{1 1}Department of Physiology and Biophysics, Weill Cornell Medical College We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

• Biology

  
  A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis Amir Rikin¹, Gabriel E. Rosenfeld¹, Kellie McCartin¹, Todd Evans^{1 1}Department of Surgery, Weill Cornell Medical College of Cornell University Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

• Immunology and Infection

  
  Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants Suma Chakravarthy¹, André C. Velásquez^1,2, Gregory B. Martin^{1,2 1}Boyce Thompson Institute for Plant Research, ²Plant Pathology and Plant-Microbe Biology, Cornell University A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

• Biology

  
  Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure Kenneth K. Kwong^1,2, Lenuta Kloetzer^1,2,3,4, Kelvin K. Wong^5,6, Jia-Qian Ren^1,2, Braden Kuo^1,2,3,4, Yan Jiang⁷, Y. Iris Chen^1,2, Suk-Tak Chan^1,2,8, Geoffrey S. Young⁹, Stephen T.C. Wong^{5,6 1}Department of Radiology, Massachusetts General Hospital, Harvard Medical School, ²Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, ³Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, ⁴Department of Medicine, Massachusetts General Hospital, Harvard Medical School, ⁵Center for biotechnology and Informatics, The Methodist Hospital Research Institute, ⁶Department of Radiology, The Methodist Hospital, Weill Cornell Medical College, ⁷Bejing University of Chinese Medicine, ⁸Department of Health Technology and Informatics, The Hong Kong Polytechnic University, ⁹Department of Radiology, Brigham and Women's Hospital, Harvard Medical School Gua Sha, traditional Chinese therapeutic skin scraping, causes subcutaneous microvascular blood extravasation. We report a protocol of bioluminescence imaging of HO-1-luciferase transgenic mice to demonstrate that Gua Sha upregulates heme oxygenase-1 (HO-1) in multiple organs.

• Biology

  
  Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana Zhiyang Zhai¹, Ha-il Jung¹, Olena K. Vatamaniuk^{1 1}Crop and Soil Sciences, Cornell University This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

• Biology

  
  Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis Xingshan Jiang¹, David Stern^{1 1}Boyce Thompson Institute for Plant Research, Cornell University Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

• Biology

  
  Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato Andrá C. Velásquez^1,2, Suma Chakravarthy², Gregory B. Martin^{1,2 1}Plant Pathology and Plant-Microbe Biology, Cornell University, ²Boyce Thompson Institute for Plant Research Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

• Biology

  
  Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties Helgi Ingolfson¹, Ruchi Kapoor², Shemille A. Collingwood², Olaf Sparre Andersen^{2 1}Department of Physiology and Biophysics, Weill Cornell Medical College, ²Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

• Biology

  
  Preparation of Artificial Bilayers for Electrophysiology Experiments Ruchi Kapoor¹, Jung H. Kim¹, Helgi Ingolfson¹, Olaf Sparre Andersen^{1 1}Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

• Biology

  
  Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment John Sawyer Ramsey¹, Georg Jander^{1 1}Boyce Thompson Institute for Plant Research, Cornell University In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

• Biology

  
  Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection Cecilia Tamborindeguy¹, Stewart Gray¹, Georg Jander^{2 1}Plant Pathology, Cornell University, ²Boyce Thompson Institute for Plant Research, Cornell University Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

• Biology

  
  Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects Martin De Vos¹, Georg Jander^{1 1}Boyce Thompson Institute for Plant Research, Cornell University Plant resistance to chewing insect herbivores can be tested in several ways. Here, we demonstrate how to set-up a choice and a no-choice experiment with the model plant Arabidopsis thaliana to identify resistance against the pest species Pieris rapae.

• Biology

  
  Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device Kevin Wong¹, Angel Ayuso-Sacido^2,3, Patrick Ahyow¹, Andrew Darling⁴, John A. Boockvar², Mingming Wu^{4 1}Biomedical Engineering Department, Cornell University, ²Neurosurgical Laboratory for Translational Stem Cell Research, Weill Cornell Brain Tumor Center, Weill Cornell Medical College of Cornell University, ³Cell Morphology Department, Instituto de Investigacion Principe Felipe, ⁴Department of Chemical and Biomolecular Engineering, Cornell University We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.