Washington University in St. Louis

53 articles published in JoVE

• Cancer Research

  
  Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays Lillian van Biljon¹, Bisiayo Fashemi¹, Jeimmy Rodriguez¹, Olivia Graham¹, Amanda Compadre¹, Katherine Fuh^1,2, Dineo Khabele¹, Mary Mullen^{1 1}Washington University in St. Louis, ²University of California San Francisco The present protocol describes methods for evaluating DNA damage repair proteins in patient-derived ovarian cancer organoids. Included here are comprehensive plating and staining methods, as well as detailed, objective quantification procedures.

• Cancer Research

  
  Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids Olivia Graham*¹, Jeimmy Rodriguez*¹, Lillian van Biljon¹, Bisiayo Fashemi¹, Emily Graham¹, Katherine Fuh^1,2, Dineo Khabele¹, Mary Mullen^{1 1}Washington University in St. Louis, ²University of California San Francisco Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

• Bioengineering

  
  Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function Elena Gracheva¹, Fei Wang¹, Abigail Matt¹, Hongwu Liang¹, Matthew Fishman^1,2, Chao Zhou^{1 1}Department of Biomedical Engineering, Washington University in St. Louis, ²Department of Computer Science and Engineering, Washington University in St. Louis The present protocol describes the generation of Drosophila melanogaster expressing eNpHR2.0 or ReaChR opsins in the heart for OCT imaging and optogenetic heart pacing. Detailed instructions for Drosophila OCT imaging and heart beating modulation, including the simulation of restorable heart arrest, bradycardia, and tachycardia in live animals at different developmental stages, are reported.

• Biology

  
  Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies Soma S. Singareddy¹, Conor McClenaghan¹, Helen I. Roessler², Robert Tryon¹, Colin G. Nichols^{1 1}Department of Cell Biology and Physiology and Center for the Investigation of Membrane Excitability Diseases, Washington University in St. Louis, ²Department of Genetics, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

• Genetics

  
  Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay Davi J. Martins*¹, Stephanie Tirman*², Annabel Quinet², Carlos F. M. Menck^{1 1}Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, ²Division of Oncology, Department of Internal Medicine, Washington University in St. Louis Here we describe two modifications of the DNA fiber assay to investigate single-stranded DNA gaps in replicating DNA after lesion induction. The S1 fiber assay enables the detection of post-replicative gaps using the ssDNA-specific S1 endonuclease, while the gap-filling assay allows visualization and quantification of gap repair.

• Neuroscience

  
  Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings Kristen N. Reikersdorfer*¹, Andrea K. Stacy*², David A. Bressler², Lauren S. Hayashi², Keith B. Hengen¹, Stephen D. Van Hooser^{2 1}Department of Biology, Washington University in St. Louis, ²Department of Biology, Program in Neuroscience, Brandeis University This protocol describes a procedure for constructing carbon fiber microelectrode arrays for chronic and acute in vivo electrophysiological recordings in mouse (Mus musculus) and ferret (Mustela putorius furo) from multiple brain regions. Each step, following the purchase of raw carbon fibers to microelectrode array implantation, is described in detail, with emphasis on microelectrode array construction.

• Chemistry

  
  Comparison of Two Different Synthesis Methods of Single Crystals of Superconducting Uranium Ditelluride Sheng Ran^1,2,3, I-Lin Liu^1,2, Shanta R. Saha^1,2, Prathum Saraf¹, Johnpierre Paglione^1,2, Nicholas P. Butch^{1,2 1}Maryland Quantum Materials Center, Department of Physics, University of Maryland, ²National Institute of Standards and Technology, ³Department of Physics, Washington University in St. Louis Here, we present a protocol to synthesize two types of UTe2 crystals: those exhibiting robust superconductivity, via chemical vapor transport synthesis, and those lacking superconductivity, via molten metal flux synthesis.

• Chemistry

  
  NMR-Based Activity Assays for Determining Compound Inhibition, IC₅₀ Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases Brian J. Stockman¹, Abinash Kaur¹, Julia K. Persaud¹, Maham Mahmood¹, Samantha F. Thuilot¹, Melissa B. Emilcar¹, Madison Canestrari¹, Juliana A. Gonzalez¹, Shannon Auletta¹, Vital Sapojnikov¹, Wagma Caravan^1,2, Samantha N. Muellers^{1,3 1}Department of Chemistry, Adelphi University, ²Department of Chemistry, Washington University in St. Louis, ³Department of Chemistry, Boston University NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 μM and 250 μM, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

• Biology

  
  Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis Jeffrey R. Millman^1,2, Teresa Doggett³, Christina Thebeau³, Sheng Zhang³, Clay F. Semenkovich¹, Rithwick Rajagopal^{3 1}Division of Metabolism, Endocrinology and Lipid Research, Department of Medicine, Washington University School of Medicine, ²Department of Biomedical Engineering, Washington University in Saint Louis, ³Department of Ophthalmology and Visual Science, Washington University School of Medicine This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

• Developmental Biology

  
  Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU Zuzana Kocsisova^1,2, Ariz Mohammad², Kerry Kornfeld¹, Tim Schedl^{2 1}Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri, ²Department of Genetics, Washington University School of Medicine, St. Louis, Missouri An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

• Chemistry

  
  Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F⁻ Justin Lyle¹, Sudharson Ravishankar Chandramoulee¹, C. Annie Hart¹, Richard Mabbs^{1 1}Department of Chemistry, Washington University in St. Louis Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

• Medicine

  
  Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation Wyatt E. Lanik¹, Lily Xu², Cliff J. Luke¹, Elise Z. Hu², Pranjal Agrawal², Victoria S. Liu², Rajesh Kumar¹, Alexa M. Bolock¹, Congrong Ma³, Misty Good^{1 1}Division of Newborn Medicine, Department of Pediatrics, Washington University School of Medicine, ²Washington University, ³Division of Newborn Medicine, Department of Pediatrics, University of Pittsburgh School of Medicine This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice.

• Developmental Biology

  
  Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells Carissa Dege¹, Christopher M. Sturgeon^{1 1}Department of Internal Medicine, Hematology Division, Washington University in St. Louis Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34⁺ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

• Bioengineering

  
  An In Vitro Organ Culture Model of the Murine Intervertebral Disc Jennifer W. Liu^1,3, Kevin H. Lin², Christian Weber¹, Sameer Bhalla², Sean Kelso⁴, Kaixi Wang³, Simon Y. Tang^{1,3,4 1}Department of Biomedical Engineering, Washington University in St. Louis, ²Department of Biology, Washington University in St. Louis, ³Department of Orthopaedic Surgery, Washington University in St. Louis, ⁴Department of Materials Science and Mechanical Engineering, Washington University in St. Louis Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

• Neuroscience

  
  Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera Eirik Søvik*^1,2, Jenny A. Plath*^3,4, Jean-Marc Devaud⁵, Andrew B. Barron^{3 1}Department of Science and Mathematics, Volda University College, ²Department of Biology, Washington University in St. Louis, ³Department of Biological Sciences, Macquarie University, ⁴Department of Biology, University of Konstanz, ⁵Research Center on Animal Cognition, CNRS, Universite de Toulouse This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

• Biology

  
  Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging Tyler M. Bauman^1,2, Emily A. Ricke², Sally A. Drew³, Wei Huang^3,4, William A. Ricke^{2,4 1}Division of Urologic Surgery, Washington University in St. Louis School of Medicine, ²Department of Urology, University of Wisconsin School of Medicine and Public Health, ³Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, ⁴O’Brien Urology Research Center, University of Wisconsin School of Medicine and Public Health Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

• Biology

  
  Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner David Razafsky¹, Chloe Potter¹, Didier Hodzic^{1 1}Department of Ophthalmology and Visual Sciences, Washington University in St. Louis School of Medicine Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

• Neuroscience

  
  Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System Frans Vinberg¹, Vladimir Kefalov^{1 1}Department of Ophthalmology and Visual Sciences, Washington University in St. Louis Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

• Medicine

  
  Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo Cara C. Rada¹, Stephanie L. Pierce², Chad A. Grotegut², Sarah K. England^{1 1}Department of Obstetrics and Gynecology, Washington University in St. Louis, ²Department of Obstetrics and Gynecology, Duke University This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

• Neuroscience

  
  A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice Daniel S. Brenner^1,2, Judith P. Golden², Sherri K. Vogt², Robert W. Gereau IV^{2 1}MSTP, Neuroscience Program, Washington University in St. Louis, ²Washington University Pain Center, Department of Anesthesiology, Washington University in St. Louis The Cold Plantar Assay (CPA) measures cold responsiveness between 30 °C and 5 °C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

• Bioengineering

  
  Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism Sina Mossahebi^2,5, Simeng Zhu^2,5, Howard Chen^1,5, Leonid Shmuylovich^3,5, Erina Ghosh^1,5, Sándor J. Kovács^{4,5 1}Department of Biomedical Engineering, Washington University in St. Louis, ²Department of Physics, Washington University in St. Louis, ³Division of Biology and Biomedical Sciences, Washington University in St. Louis, ⁴Department of Medicine, Cardiovascular Division, Washington University in St. Louis, ⁵Cardiovascular Biophysics Lab, Washington University in St. Louis Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation, and load parameters and elucidates 'new' physiology while providing sensitive and specific indexes of dysfunction.

• Neuroscience

  
  Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb Wayne I. Doyle¹, Gary F. Hammen², Julian P. Meeks^{1 1}Department of Neuroscience, UT Southwestern Medical Center, ²Department of Anatomy and Neurobiology, Washington University in St. Louis The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

• Biology

  
  The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry Michael W. Rudokas¹, Zoltan Varga¹, Angela R. Schubert¹, Alexandra B. Asaro¹, Jonathan R. Silva^{1 1}Department of Biomedical Engineering, Washington University in St. Louis The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

• Immunology and Infection

  
  Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium Megan E. Lau¹, David A. Hunstad^{1,2 1}Department of Pediatrics, Washington University School of Medicine, ²Department of Molecular Microbiology, Washington University School of Medicine We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.

• Environment

  
  Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations Lian He¹, Amelia B Chen^1,2, Yi Yu², Leah Kucera³, Yinjie Tang^{1 1}Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, ²School of Pharmaceutical Sciences, Wuhan University of China, ³Department of Earth and Planetary Sciences, Washington University in St. Louis Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype "flue gas to algal cultivation" systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

• Neuroscience

  
  Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo Ariel M. Lyons-Warren¹, Tsunehiko Kohashi^1,2, Steven Mennerick³, Bruce A. Carlson^{1 1}Department of Biology, Washington University in St. Louis, ²Division of Biological Science, Graduate School of Science, Nagoya University, ³Department of Psychiatry, Washington University in St. Louis Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

• Neuroscience

  
  Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System Sarah L. DeVos¹, Timothy M. Miller^{1 1}Department of Neurology, Washington University in St. Louis School of Medicine We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

• Neuroscience

  
  Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits Debajit Saha¹, Kevin Leong¹, Nalin Katta¹, Baranidharan Raman^{1 1}Department of Biomedical Engineering, Washington University in St. Louis We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

• Medicine

  
  Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS) Ameet I. Thaker¹, Anisa Shaker¹, M. Suprada Rao¹, Matthew A. Ciorba^{1 1}Division of Gastroenterology, Washington University School of Medicine We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

• Neuroscience

  
  Recording Human Electrocorticographic (ECoG) Signals for Neuroscientific Research and Real-time Functional Cortical Mapping N. Jeremy Hill¹, Disha Gupta^1,2, Peter Brunner^1,2, Aysegul Gunduz^1,2, Matthew A. Adamo³, Anthony Ritaccio², Gerwin Schalk^{1,2,4,5,6,7 1}Wadsworth Center, New York State Department of Health, ²Department of Neurology, Albany Medical College, ³Department of Neurosurgery, Albany Medical College, ⁴Department of Neurosurgery, Washington University, ⁵Department of Biomed. Eng., Rensselaer Polytechnic Institute, ⁶Department of Biomed. Sci., State University of New York at Albany, ⁷Department of Elec. and Comp. Eng., University of Texas at El Paso We present a method for collecting electrocorticographic signals for research purposes from humans who are undergoing invasive epilepsy monitoring. We show how to use the BCI2000 software platform for data collection, signal processing and stimulus presentation. Specifically, we demonstrate SIGFRIED, a BCI2000-based tool for real-time functional brain mapping.

• Biology

  
  Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER Francesco Vallania¹, Enrique Ramos¹, Sharon Cresci², Robi D. Mitra¹, Todd E. Druley^{1,3 1}Center for Genome Sciences and Systems Biology, Department of Genetics, Washington University School of Medicine, ²Department of Internal Medicine, Washington University School of Medicine, ³Department of Pediatrics, Washington University School of Medicine Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

• Neuroscience

  
  Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System Mingjie Li¹, Nada Husic¹, Ying Lin¹, B. Joy Snider^{1 1}Department of Neurology and Hope Center for Neurological Disorders, Washington University School of Medicine In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

• Neuroscience

  
  Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses Alexander V. Kolesnikov¹, Vladimir J. Kefalov^{1 1}Department of Ophthalmology and Visual Sciences, Washington University School of Medicine We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

• Bioengineering

  
  Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice Victoria P. Le¹, Attila Kovacs², Jessica E. Wagenseil^{1 1}Department of Biomedical Engineering, Saint Louis University, ²Department of Internal Medicine, Washington University School of Medicine Measuring left ventricular pressure (LV) in embryonic and neonatal mice is described. Pressure is measured by inserting a needle connected to a fluid-filled transducer into the LV under ultrasound guidance. Care must be taken to maintain normal cardiac function during the experimental protocol.

• Biology

  
  Metabolic Pathway Confirmation and Discovery Through ¹³C-labeling of Proteinogenic Amino Acids Le You¹, Lawrence Page², Xueyang Feng¹, Bert Berla¹, Himadri B. Pakrasi³, Yinjie J. Tang^{1 1}Department of Energy, Environmental and Chemical Engineering, Washington University, ²Department of Biology, Washington University, ³Department of Energy, Environmental and Chemical Engineering and Department of Biology, Washington University ¹³C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

• Immunology and Infection

  
  Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging Joel R. Meyerson^1,2, Tommi A. White¹, Donald Bliss³, Amy Moran³, Alberto Bartesaghi¹, Mario J. Borgnia¹, M. Jason V. de la Cruz¹, David Schauder¹, Lisa M. Hartnell¹, Rachna Nandwani^1,4, Moez Dawood⁵, Brianna Kim⁶, Jun Hong Kim⁷, John Sununu⁸, Lisa Yang⁹, Siddhant Bhatia¹⁰, Carolyn Subramaniam¹, Darrell E. Hurt¹¹, Laurent Gaudreault¹², Sriram Subramaniam^{1 1}Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, ²The Medical Research Council Mitochondrial Biology Unit, University of Cambridge, ³National Library of Medicine, National Institutes of Health, ⁴Massachusetts Institute of Technology, ⁵William Fremd High School, ⁶University of Virginia, ⁷Duke University, ⁸Yale University, ⁹University of Notre Dame, ¹⁰Washington University in St. Louis, ¹¹Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, ¹²Thomas Jefferson High School for Science and Technology The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

• Biology

  
  Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo Benjamen A. Filas¹, Victor D. Varner¹, Dmitry A. Voronov^1,2, Larry A. Taber^{1,3 1}Department of Biomedical Engineering, Washington University, ²Institute for Information Transmission Problems, Russian Academy of Sciences, ³Department of Mechanical Engineering and Materials Science, Washington University This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

• Bioengineering

  
  Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart Di Lang¹, Matthew Sulkin¹, Qing Lou¹, Igor R. Efimov^{1 1}Department of Biomedical Engineering, Washington University in St. Louis This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

• Bioengineering

  
  Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart Qing Lou¹, Wenwen Li¹, Igor R. Efimov^{1 1}Department of Biomedical Engineering, Washington University in St. Louis This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

• Bioengineering

  
  Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement Dawn M. Johnson¹, Natalie A. LaFranzo¹, Joshua A. Maurer^{1 1}Department of Chemistry, Washington University in St. Louis Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

• Neuroscience

  
  Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation Cynthia L. Montana¹, Connie A. Myers¹, Joseph C. Corbo^{1 1}Department of Pathology and Immunology, Washington University School of Medicine This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

• Bioengineering

  
  Three-dimensional Optical-resolution Photoacoustic Microscopy Song Hu¹, Konstantin Maslov¹, Lihong V. Wang^{1 1}Optical Imaging Laboratory, Department of Biomedical Engineering, Washington University in St. Louis Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

• Biology

  
  Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes Junqiu Yang¹, Kelli Delaloye², Urvi S. Lee², Jianmin Cui^{3 1}Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, ²Department of Biomedical Engineering, Washington University in St. Louis, ³Department of Biomedical Engineering and Cardiac Bioelectricity and Arrhythmia Center, Washington University in St. Louis Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

• Immunology and Infection

  
  Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells Nayan J. Sarma¹, Akiko Takeda¹, Nabeel R. Yaseen^{1 1}Department of Pathology and Immunology, Washington University School of Medicine The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

• Immunology and Infection

  
  Non-invasive Imaging of Leukocyte Homing and Migration in vivo Baomei Wang¹, Bernd H. Zinselmeyer^1,2, Jeremiah R. McDole¹, Peggy A. Gieselman¹, Mark J. Miller^{1 1}Department of Pathology and Immunology, Washington University in St. Louis, ²National Institute of Neurological Disorders and Stroke, NINDS, NIH - National Institute of Health Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

• Medicine

  
  Technique to Collect Fungiform (Taste) Papillae from Human Tongue Andrew I. Spielman*¹, M. Yanina Pepino², Roy Feldman^3,4,5, Joseph G. Brand*^{4,6 1}Department 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.

• Biology

  
  Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation Enrique Amaya¹, Kristen Kroll^{2 1}The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, ²Department of Developmental Biology, Washington University School of Medicine This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

• Immunology and Infection

  
  In vivo Imaging of Transgenic Leishmania Parasites in a Live Host Colin J. Thalhofer¹, Joel W. Graff², Laurie Love-Homan³, Suzanne M. Hickerson⁴, Noah Craft⁵, Stephen M. Beverley⁴, Mary E. Wilson^{6,7 1}Interdisciplinary Immunology Program, University of Iowa, and the VA Medical Center, ²Department of Biochemistry, University of Iowa, and the VA Medical Center, ³Department of Internal Medicine, University of Iowa, ⁴Department of Molecular Microbiology, Washington University School of Medicine, ⁵Division of Dermatology, Harbor-UCLA Medical Center, Hanley-Hardison Research Center, ⁶Interdisciplinary Immunology Program, Iowa City VA Medical Center, ⁷Departments of Internal Medicine, Microbiology and Epidemiology, University of Iowa An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

• Biology

  
  Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System Amos Danielli^1,2, Noga Porat³, Marcelo Ehrlich⁴, Ady Arie^{1 1}Department of Physical Electronics, Faculty of Engineering, Tel Aviv University, ²Department of Biomedical Engineering, Washington University in St. Louis, ³Department of Biological Sciences, University of Illinois, ⁴Department of Cell Research and Immunology, Tel Aviv University Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

• Neuroscience

  
  Multielectrode Array Recordings of the Vomeronasal Epithelium Hannah A. Arnson¹, Xiaoyan Fu¹, Timothy E. Holy^{1 1}Anatomy and Neurobiology, Washington University School of Medicine Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

• Biology

  
  Single-cell Suction Recordings from Mouse Cone Photoreceptors Jin-Shan Wang¹, Vladimir J Kefalov^{1 1}Department of Ophthalmology and Visual Sciences, Washington University in St. Louis, School of Medicine We will show how to record flash responses from single mouse cones using a suction electrode.

• Biology

  
  Presynaptically Silent Synapses Studied with Light Microscopy Krista L. Moulder¹, Xiaoping Jiang¹, Amanda A. Taylor¹, Ann M. Benz¹, Steven Mennerick^{1,2,3 1}Department of Psychiatry, Washington University School of Medicine, ²Department of Anatomy, Washington University School of Medicine, ³Department of Neurobiology, Washington University School of Medicine Glutamatergic synapses can switch from an active mode to a silent mode. We demonstrate that presynaptic activity status in dissociated culture of rodent neurons is visualized using a fixable form of the FM1-43 dye to visualize active synapses and immunostaining with vGluT-1 antibody to visualize all glutamate synapses.

• Biology

  
  Pyrosequencing: A Simple Method for Accurate Genotyping Cristi King¹, Tiffany Scott-Horton^{1 1}Department of Internal Medicine, Washington University in St. Louis Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.