Washington University in St. Louis View Institution's Website 54 articles published in JoVE Biology Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration Jacob C. Parson1, Nicole K. Biltz1, Gretchen A. Meyer1,2 1Program in Physical Therapy, Washington University in St. Louis, 2Departments of Neurology, Orthopaedic Surgery and Biomedical Engineering, Washington University in St. Louis The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT. 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 Fuh1,2, Dineo Khabele1, Mary Mullen1 1Washington 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 Fuh1,2, Dineo Khabele1, Mary Mullen1 1Washington 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 Fishman1,2, Chao Zhou1 1Department 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. Nichols1 1Department 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. Menck1 1Department 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 Hooser2 1Department 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 Ran1,2,3, I-Lin Liu1,2, Shanta R. Saha1,2, Prathum Saraf1, Johnpierre Paglione1,2, Nicholas P. Butch1,2 1Maryland 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 Caravan1,2, Samantha N. Muellers1,3 1Department 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. Millman1,2, Teresa Doggett3, Christina Thebeau3, Sheng Zhang3, Clay F. Semenkovich1, Rithwick Rajagopal3 1Division 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 Kocsisova1,2, Ariz Mohammad2, Kerry Kornfeld1, Tim Schedl2 1Department 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 Mabbs1 1Department 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 Good1 1Division 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. Sturgeon1 1Department 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. Liu1,3, Kevin H. Lin2, Christian Weber1, Sameer Bhalla2, Sean Kelso4, Kaixi Wang3, Simon Y. Tang1,3,4 1Department 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. Barron3 1Department 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. Bauman1,2, Emily A. Ricke2, Sally A. Drew3, Wei Huang3,4, William A. Ricke2,4 1Division 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 Hodzic1 1Department 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 Kefalov1 1Department 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. England1 1Department 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. Brenner1,2, Judith P. Golden2, Sherri K. Vogt2, Robert W. Gereau IV2 1MSTP, 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 Mossahebi2,5, Simeng Zhu2,5, Howard Chen1,5, Leonid Shmuylovich3,5, Erina Ghosh1,5, Sándor J. Kovács4,5 1Department 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. Meeks1 1Department 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. Silva1 1Department 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. Hunstad1,2 1Department 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 Chen1,2, Yi Yu2, Leah Kucera3, Yinjie Tang1 1Department 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 Kohashi1,2, Steven Mennerick3, Bruce A. Carlson1 1Department 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. Miller1 1Department 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 Raman1 1Department 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. Ciorba1 1Division 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 Gupta1,2, Peter Brunner1,2, Aysegul Gunduz1,2, Matthew A. Adamo3, Anthony Ritaccio2, Gerwin Schalk1,2,4,5,6,7 1Wadsworth 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. Druley1,3 1Center 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 Snider1 1Department 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. Kefalov1 1Department 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. Wagenseil1 1Department 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. Tang1 1Department 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. Meyerson1,2, Tommi A. White1, Donald Bliss3, Amy Moran3, Alberto Bartesaghi1, Mario J. Borgnia1, M. Jason V. de la Cruz1, David Schauder1, Lisa M. Hartnell1, Rachna Nandwani1,4, Moez Dawood5, Brianna Kim6, Jun Hong Kim7, John Sununu8, Lisa Yang9, Siddhant Bhatia10, Carolyn Subramaniam1, Darrell E. Hurt11, Laurent Gaudreault12, Sriram Subramaniam1 1Laboratory 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. Voronov1,2, Larry A. Taber1,3 1Department 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. Efimov1 1Department 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. Efimov1 1Department 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. Maurer1 1Department 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. Corbo1 1Department 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. Wang1 1Optical 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 Cui3 1Department 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. Yaseen1 1Department 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. Zinselmeyer1,2, Jeremiah R. McDole1, Peggy A. Gieselman1, Mark J. Miller1 1Department 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 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. Biology Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation Enrique Amaya1, Kristen Kroll2 1The 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. Wilson6,7 1Interdisciplinary 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 Danielli1,2, Noga Porat3, Marcelo Ehrlich4, Ady Arie1 1Department 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. Holy1 1Anatomy 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 Kefalov1 1Department 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 Mennerick1,2,3 1Department 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-Horton1 1Department 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.