Vanderbilt University View Institution's Website 70 articles published in JoVE Biology Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes Adeline E. Williams1,2, Irma Sanchez-Vargas1, Lindsay E. Martin2,3, Ines Martin-Martin2,4, Susi Bennett1, Ken E. Olson1, Eric Calvo2 1Center for Vector-borne Infectious Diseases, Department of Microbiology, Immunology, and Pathology, Colorado State University, 2Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 3Department of Biological Sciences, Vanderbilt University, 4National Center for Microbiology, Instituto de Salud Carlos III The present protocol describes how to measure common life parameter data in Aedes aegypti mosquitoes, including fecundity, wing size, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These measurements can be used to assess the fitness of transgenic mosquitoes. Immunology and Infection Murine Fecal Isolation and Microbiota Transplantation Jeanne A. Ishimwe1, Jianyong Zhong2,3, Valentina Kon2, Annet Kirabo1 1Division of Clinical Pharmacology, Department of Medicine Vanderbilt University Medical Center and Department of Molecular Physiology and Biophysics, Vanderbilt University, 2Division of Pediatric Nephrology Department of Pediatrics, Vanderbilt University Medical Center, 3Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center The goal here is to outline a protocol to investigate the mechanisms of dysbiosis in cardiovascular disease. This paper discusses how to aseptically collect and transplant murine fecal samples, isolate intestines, and use the "Swiss-roll" method, followed by immunostaining techniques to interrogate changes in the gastrointestinal tract. Medicine Pediatric Animal Model of Extracorporeal Cardiopulmonary Resuscitation After Prolonged Circulatory Arrest Madeleine Ball*1,2, Sergio Benito*1,3, Juliana Pilar Caride1, Cristina Ruiz-Herguido1, Marta Camprubí-Camprubí1,4, Joan Sanchez-de-Toledo1,5,6 1Sant Joan de Déu Research Institute, 2Vanderbilt University School of Medicine, Vanderbilt University, 3Department of Pediatric Critical Care Medicine, Sant Joan de Déu Hospital, 4BCNatal-Barcelona Center for Maternal Fetal and Neonatal Medicine, Sant Joan de Déu Hospital, 5Department of Pediatric Cardiology, Sant Joan de Déu Hospital, 6Department of Critical Care Medicine, University of Pittsburgh This protocol describes a neonatal porcine model of cardiopulmonary bypass (CPB), with circulatory and cardiac arrest as a tool for studying severe brain damage and other complications secondary to CPB. Medicine A Heterotopic Mouse Model for Studying Laryngeal Transplantation Maeve M. Kennedy*1, Egehan Salepci*1,2, Cheryl Myers1, Marshall Strome3,4, David G. Lott1,5 1Head and Neck Regenerative Medicine Laboratory, Center for Regenerative Medicine, Mayo Clinic Arizona, 2Department of Otorhinolaryngology, University of Health Sciences, Sisli Hamidiye Etfal Training and Research Hospital, 3Department of Otolaryngology, Vanderbilt University, 4Cleveland Clinic Head and Neck Institute, 5Division of Laryngology, Department of Otolaryngology Head and Neck Surgery, Mayo Clinic Arizona The aim of this manuscript is to describe the microsurgical steps required to perform a heterotopic laryngeal transplant in mice. The advantages of this mouse model compared to other animal models of laryngeal transplantation are its cost-effectiveness and the availability of immunologic assays and data. Biology Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum James S. White1,2, Kimberly S. LaFever2, Andrea Page-McCaw1,2 1Dept. Cell and Developmental Biology, Vanderbilt University, 2Program in Developmental Biology, Vanderbilt University The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article. Biology Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics Shobana Jayapalan1,2, Ananya Nandy1,2, Elizabeth Rendina-Ruedy1,2,3 1Vanderbilt Center for Bone Biology, Division of Clinical Pharmacology, Vanderbilt University Medical Center, 2Department of Medicine, Division of Clinical Pharmacology, Vanderbilt University Medical Center, 3Department of Molecular Physiology and Biophysics, Vanderbilt University Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status. Biology Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro Zhu Li1, Matthew J. W. Hampton1, Matthew B. Barajas1, Matthias L. Riess1,2,3 1Department of Anesthesiology, Vanderbilt University Medical Center, 2Anesthesiology, TVHS VA Medical Center, 3Department of Pharmacology, Vanderbilt University Spatial distance is a key parameter in assessing hypoxia/reoxygenation injury in a co-culture model of separate endothelial and cardiomyocyte cell layers, suggesting, for the first time, that optimizing the co-culture spatial environment is necessary to provide a favorable in vitro model for testing the role of endothelial cells in cardiomyocyte protection. Neuroscience Imaging Dendritic Spines in Caenorhabditis elegans Andrea Cuentas-Condori1, D. M. Miller III1,2 1Department of Cell and Developmental Biology, Vanderbilt University, 2Program of Neuroscience, Vanderbilt University Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans. These approaches support the development of mutant screens for genes that define dendritic spine shape or function. Bioengineering A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep Rei Ukita1, John W. Stokes1, W. Kelly Wu1, Jennifer Talackine1, Nancy Cardwell1, Yatrik Patel1, Clayne Benson5, Caitlin T. Demarest1, Erika B. Rosenzweig3, Keith Cook2, Emily J. Tsai4, Matthew Bacchetta1,6 1Department of Thoracic Surgery, Vanderbilt University Medical Center, 2Department of Biomedical Engineering, Carnegie Mellon University, 3Department of Pediatrics, Columbia University Medical Center, Columbia University Irving Medical Center, 4Department of Medicine, Columbia University Medical Center, Columbia University Irving Medical Center, 5Department of Anesthesiology, Vanderbilt University Medical Center, 6Department of Biomedical Engineering, Vanderbilt University This manuscript describes the surgical technique and experimental approach to develop severe right ventricular pressure overload to model their adaptive and maladaptive phenotypes. Biology Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence Cassandra Warden*1, Alan J. Simmons*2, Lejla Pasic3, Ashley Pitzer4,6, Sean S. Davies4, Justin H. Layer5, Raymond L. Mernaugh3, Annet Kirabo4,6 1Vanderbilt Eye Institute, Vanderbilt University Medical Center, 2Department of Cell and Developmental Biology, Vanderbilt University, 3Department of Biochemistry, Vanderbilt University, 4Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, 5Division of Hematology and Oncology, Indiana University School of Medicine, 6Department of Molecular Physiology and Biophysics, Vanderbilt University This article provides a detailed methodology for the measurement of isolevuglandins in tissues by immunofluorescence using alkaline phosphatase-conjugated ScFv D11 antibody. Hypertension models in both mice and humans are used to explain the step-by-step procedures and fundamental principles associated with isolevuglandin measurement in tissue samples. Bioengineering Low-Cost, Volume-Controlled Dipstick Urinalysis for Home-Testing Emily Kight1, Iftak Hussain1, Audrey K. Bowden1,2 1Vanderbilt Biophotonics Center and Department of Biomedical Engineering, Vanderbilt University, 2Department of Electrical Engineering and Computer Science, Vanderbilt University Dipstick urinalysis is a quick and affordable method of assessing one’s personal state of health. We present a method to perform accurate, low-cost dipstick urinalysis that removes the primary sources of error associated with traditional dip-and-wipe protocols and is simple enough to be performed by lay users at home. Chemistry Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT) Albert B. Arul1, Renã A.S. Robinson1,2,3,4 1Department of Chemistry, Vanderbilt University, 2Vanderbilt Memory & Alzheimer's Center, Vanderbilt University Medical Center, 3Vanderbilt Brain Institute, Vanderbilt University Medical Center, 4Vanderbilt Institute of Chemical Biology, Vanderbilt University Medical Center Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced sample multiplexing strategy that is capable of increasing the number of samples that can be analyzed simultaneously with available isobaric tags. Incorporation of a robotic platform has greatly increased experimental throughput, reproducibility, and quantitative accuracy. Medicine A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development Brian A. Juber1, Timothy G. Elgin1, Erin M. Fricke2, Huyiu Gong1, Jeffrey Reese3, Steven J. McElroy1,4 1Division of Neonatology, Stead Family Department of Pediatrics, University of Iowa, 2Division of Maternal Fetal Medicine, Department of Obstetrics & Gynecology, University of Iowa, 3Division of Neonatology, Department of Pediatrics, Vanderbilt University, 4Department of Microbiology & Immunology, University of Iowa We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring’s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis. Developmental Biology Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption Joseph M. Elsakr1, Charles Deeter2, Valerie Ricciardi3, Maureen Gannon1,4,5,6 1Department of Molecular Physiology and Biophysics, Vanderbilt University, 2Agilent Technologies, 3Department of Biological Sciences, Vanderbilt University, 4Department of Veterans Affairs Tennessee Valley, 5Department of Medicine, Vanderbilt University Medical Center, 6Department of Cell and Developmental Biology, Vanderbilt University This protocol demonstrates the accurate and reproducible measurement of oxygen consumption in non-human primate pancreatic islets. The islet loading techniques and coating of the microplate provide a framework for efficient measurement of respiration in other types of cultured spheroids. Cancer Research Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels Steven M Alves1, Tian Zhu1, Anastasia Shostak1, Ninna S. Rossen2, Marjan Rafat1,3,4 1Department of Chemical and Biomolecular Engineering, Vanderbilt University, 2Department of Radiation Oncology, Stanford University, 3Depattment of Biomedical Engineering, Vanderbilt University, 4Department of Radiation Oncology, Vanderbilt University Medical Center This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation. Bioengineering Growth and Characterization of Irradiated Organoids from Mammary Glands Benjamin C. Hacker1, Javier D. Gomez1, Carlos A. Silvera Batista1, Marjan Rafat1,2,3 1Department of Chemical and Biomolecular Engineering, Vanderbilt University, 2Department of Biomedical Engineering, Vanderbilt University, 3Department of Radiation Oncology, Vanderbilt University Medical Center Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue. Developmental Biology Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence Zhentao Zhang1,2,3, Young-Jae Nam1,2,3 1Department of Medicine, Division of Cardiovascular Medicine, Vanderbilt University Medical Center, 2Department of Cell and Developmental Biology, Vanderbilt University, 3Vanderbilt Center for Stem Cell Biology, Vanderbilt University We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods. Medicine Fat-Water Phantoms for Magnetic Resonance Imaging Validation: A Flexible and Scalable Protocol Emily C. Bush1, Aliya Gifford2, Crystal L. Coolbaugh1, Theodore F. Towse1,3,4, Bruce M. Damon1,5,6,7, E. Brian Welch1,5 1Vanderbilt University Institute of Imaging Science (VUIIS), Vanderbilt University Medical Center, 2Department of Biomedical Informatics, Vanderbilt University Medical Center, 3Department of Physical Medicine and Rehabilitation, Vanderbilt University Medical Center, 4Department of Biomedical Sciences, Grand Valley State University, 5Department of Radiology & Radiological Sciences, Vanderbilt University Medical Center, 6Department of Biomedical Engineering, Vanderbilt University, 7Department of Molecular Physiology and Biophysics, Vanderbilt University The purpose of this work is to describe a protocol for creating a practical fat-water phantom that can be customized to produce phantoms with varying fat percentages and volumes. Genetics Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions Stephanie S.L. Birnbaum1, David C. Rinker1, Patrick Abbot1 1Biological Sciences Department, Vanderbilt University The aphid Aphis nerii colonizes on highly-defended plants in the dogbane family (Apocyanaceae) and provides numerous opportunities to study plant-insect interactions. Here, we present a series of protocols for the maintenance of plant and aphid cultures, and the generation and analysis of molecular and -omic data for A. nerii. Neuroscience FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation Danielle L. Kopke1, Kendal Broadie2 1Department of Biological Sciences, Vanderbilt University, 2Departments of Biological Sciences, Pharmacology, Cell and Developmental Biology, Kennedy Center for Research on Human Development, Vanderbilt University and Medical Center Synaptic vesicle (SV) cycling is the core mechanism of intercellular communication at neuronal synapses. FM dye uptake and release are the primary means of quantitatively assaying SV endo- and exocytosis. Here, we compare all the stimulation methods to drive FM1-43 cycling at the Drosophila neuromuscular junction (NMJ) model synapse. Genetics Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing Ahmed Elaswad*1,2, Karim Khalil*1,3, David Cline1, Patrick Page-McCaw4, Wenbiao Chen4, Maximilian Michel5, Roger Cone5, Rex Dunham1 1School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, 2Department of Animal Wealth Development, Faculty of Veterinary Medicine, Suez Canal University, 3Anatomy and Embryology Department, Faculty of Veterinary Medicine, Cairo University, 4Department of Molecular Physiology and Biophysics, Vanderbilt University, 5Life Science Institute, University of Michigan A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes. Chemistry One-pot Microwave-assisted Conversion of Anomeric Nitrate-esters to Trichloroacetimidates D. Jamin Keith1, Stefan A. Marasligiller*1, Alexander W. Sasse*1, Steven D. Townsend1,2 1Department of Chemistry, Vanderbilt University, 2Institute of Chemical-Biology, Vanderbilt University A 2-azido-1-nitrate-ester can be converted to the corresponding 2-azido-1-trichloroacetimidate in a one-pot procedure. The goal of the manuscript is to demonstrate utility of the microwave reactor in carbohydrate synthesis. Chemistry Nanosponge Tunability in Size and Crosslinking Density Laken L. Kendrick-Williams1, Eva Harth1 1Chemistry Department, Vanderbilt University This article describes a process for tuning the size and crosslinking density of covalently crosslinked nanoparticles from linear polyesters containing pendant functionality. By tailoring synthesis parameters (polymer molecular weight, pendant functionality incorporation, and crosslinker equivalents), a desired nanoparticle size and crosslinking density can be achieved for drug delivery applications. Biochemistry Expression, Purification, and Antimicrobial Activity of S100A12 Emmanuel Jackson1, Saffron Little1, Dana S. Franklin1, Jennifer A. Gaddy2,3, Steven M. Damo1,4 1Department of Life and Physical Sciences, Fisk University, 2Tennessee Valley Healthcare Systems, U. S. Dept. of Veterans Affairs, 3Department of Medicine - Division of Infectious Diseases, Vanderbilt University Medical School, 4Department of Biochemistry, Vanderbilt University Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori. Immunology and Infection Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension Fanny Laroumanie1, Bethany L. Dale2, Mohamed A. Saleh3, Meena S. Madhur1 1Department of Medicine, Division of Clinical Pharmacology, Vanderbilt University Medical Center, 2Department of Molecular Physiology and Biophysics, Vanderbilt University, 3Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University This article provides detailed methodology to identify and quantify functional T lymphocyte subsets present within murine kidney, aorta and lymph nodes by intracellular staining and flow cytometry. The model of angiotensin II induced hypertension was chosen to explain, step-by-step, the procedures and fundamental principles of flow cytometry and intracellular staining. Biology The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision Andrea A. Perreault1, Bryan J. Venters1 1Department of Molecular Physiology and Biophysics, Vanderbilt University Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing. Medicine Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease Bruce M. Damon1,2,3,4, Ke Li1,2, Richard D. Dortch1,2, E. Brian Welch1,2, Jane H. Park1,2,4, Amanda K. W. Buck1,2, Theodore F. Towse1,2,5, Mark D. Does1,2,3, Daniel F. Gochberg1,2,6, Nathan D. Bryant1,2 1Institute of Imaging Science, Vanderbilt University, 2Department of Radiology and Radiological Sciences, Vanderbilt University, 3Department of Biomedical Engineering, Vanderbilt University, 4Department of Molecular Physiology and Biophysics, Vanderbilt University, 5Department of Physical Medicine and Rehabilitation, Vanderbilt University, 6Department of Physics and Astronomy, Vanderbilt University Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods. Bioengineering Sustained Administration of β-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres Raymond C. Pasek1, Taylor E. Kavanaugh2, Craig L. Duvall2, Maureen A. Gannon1,3 1Department of Medicine, Vanderbilt University Medical Center, 2School of Engineering, Vanderbilt University, 3Department of Veterans Affairs, Tennessee Valley Health Authority Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of β-cell proliferation. This method is suitable for determining the efficacy of candidate β-cell mitogens. Engineering Data Acquisition Protocol for Determining Embedded Sensitivity Functions Janette J. Meyer1, Douglas E. Adams1, Janene Silvers2 1Laboratory for Systems Integrity and Reliability (LASIR), Vanderbilt University, 2Mechanical Engineering, Purdue University The data acquisition procedure for determining embedded sensitivity functions is described. Data is acquired and representative results are shown for a residential scale wind turbine blade. Developmental Biology Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2 Jeffery B. Bylund1,2, Antonis K. Hatzopoulos1,3 1Department of Medicine, Division of Cardiovascular Medicine, Vanderbilt University School of Medicine, 2Department of Pharmacology, Vanderbilt University, 3Department of Cell & Developmental Biology, Vanderbilt University School of Medicine Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype. Chemistry Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II Keersten M. Davis1, Anna L. Bitting1, Christine F. Markwalter1, Westley S. Bauer1, David W. Wright1 1Department of Chemistry, Vanderbilt University Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application. Behavior Testing Sensory and Multisensory Function in Children with Autism Spectrum Disorder Sarah H. Baum1, Ryan A. Stevenson2, Mark T. Wallace3 1Vanderbilt Brain Institute, Vanderbilt University Medical Center, 2Department of Psychology, University of Toronto, 3Department of Hearing and Speech Sciences, Vanderbilt University We describe how to implement a battery of behavioral tasks to examine the processing and integration of sensory stimuli in children with ASD. The goal is to characterize individual differences in temporal processing of simple auditory and visual stimuli and relate these to higher order perceptual skills like speech perception. Medicine Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images Aliya Gifford1, Theodore F. Towse2, Ronald C. Walker3, Malcolm J. Avison4, E. Brian Welch3 1Chemical and Physical Biology Program, Vanderbilt University, 2Department of Physical Medicine and Rehabilitation, Vanderbilt University School of Medicine, 3Radiology & Radiological Sciences, Vanderbilt University Medical Center, 4Department of Pharmacology, Vanderbilt University The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 °C) and cold conditions (17 °C) in order to map brown adipose tissue (BAT) in adult human subjects. Biology Isolation and Physiological Analysis of Mouse Cardiomyocytes Gretchen M. Roth1, David M. Bader1,2, Elise R. Pfaltzgraff2 1Department of Medicine, Vanderbilt University, 2Department of Cell and Developmental Biology, Vanderbilt University Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause. Behavior Quantitative Assessment of Cortical Auditory-tactile Processing in Children with Disabilities Nathalie L. Maitre1, Alexandra P. Key2,3 1Department of Pediatrics, Monroe Carell Jr. Children's Hospital at Vanderbilt, Vanderbilt University, 2Vanderbilt Kennedy Center, Vanderbilt University, 3Department of Hearing and Speech Sciences, Vanderbilt University Objective and easy measurement of sensory processing is extremely difficult in nonverbal or vulnerable pediatric patients. We developed a new methodology to quantitatively assess infants and children's cortical processing of light touch, speech sounds, and the multisensory processing of the 2 stimuli, without requiring active subject participation or causing discomfort in vulnerable patients. Medicine Echocardiographic Assessment of the Right Heart in Mice Evan Brittain1, Niki L. Penner2, James West2, Anna Hemnes2 1Division of Cardiovascular Medicine, Vanderbilt University Medical Center, 2Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease. Medicine Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice Nataliya I. Skrypnyk1, Raymond C. Harris1, Mark P. de Caestecker1 1Division of Nephrology, Vanderbilt University Medical Center We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment. Immunology and Infection Isolation of Adipose Tissue Immune Cells Jeb S. Orr1, Arion J. Kennedy1, Alyssa H. Hasty1 1Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease. Immunology and Infection Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs Brian C. Evans*1,2, Christopher E. Nelson*1,2, Shann S. Yu*1,2, Kelsey R. Beavers2,3, Arnold J. Kim1, Hongmei Li1,2, Heather M. Nelson4, Todd D. Giorgio1,2,5,6, Craig L. Duvall1,2 1Department of Biomedical Engineering, Vanderbilt University, 2Vanderbilt Institute for Nanoscale Science & Engineering, Vanderbilt University, 3Interdisciplinary Materials Science Program, Vanderbilt University, 4Monroe Carell Jr. Children's Hospital, Vanderbilt University Medical Center, 5Department of Chemical & Biomolecular Engineering, Vanderbilt University, 6Department of Cancer Biology, Vanderbilt University A hemolysis assay can be used as a rapid, high-throughput screen of drug delivery systems' cytocompatibility and endosomolytic activity for intracellular cargo delivery. The assay measures the disruption of erythrocyte membranes as a function of environmental pH. Immunology and Infection Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source Gleb Pishchany1, Kathryn P. Haley1, Eric P. Skaar1 1Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical School Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition. Biology High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels Rene Raphemot1,2, C. David Weaver1,3, Jerod S. Denton1,2 1Department of Pharmacology, Vanderbilt University School of Medicine, 2Department of Anesthesiology, Vanderbilt University School of Medicine, 3Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented. Immunology and Infection Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice Wen-Chi Chen*1, Sung-Hyun Park*1, Carol Hoffman1, Cecil Philip1, Linda Robinson2, James West2, Gabriele Grunig1,3 1Department of Environmental Medicine, New York University School of Medicine, Tuxedo, 2Division of Allergy, Pulmonary, & Critical Care Medicine, Department of Medicine, Vanderbilt University Medical Center, 3Division of Pulmonary Medicine, New York University School of Medicine A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies. Medicine Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics Jacob A. Martin1, Alexander S. Maris1, Moneeb Ehtesham1, Robert J. Singer1 1Department of Neurological Surgery, Vanderbilt University School of Medicine Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier. Medicine 3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System Teagan J. Walter1,2, Erin E. Sparks3, Stacey S. Huppert2 1Department of Cell and Developmental Biology, Center for Stem Cell Biology, Vanderbilt University, 2Division of Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital, 3Department of Biology, Duke University A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture. Immunology and Infection Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models Wei Han1, Hui Li1, Brahm H. Segal2,3, Timothy S. Blackwell1 1Department of Medicine, Vanderbilt University School of Medicine, 2Departments of Medicine and Immunology, Roswell Park Cancer Institute, 3Department of Medicine, University at Buffalo School of Medicine NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described. Medicine Models of Bone Metastasis J. Preston Campbell1,2, Alyssa R. Merkel2,3,4, S. Kathryn Masood-Campbell2,4, Florent Elefteriou1,2,4,5, Julie A. Sterling2,3,4,5 1Department of Pharmacology, Vanderbilt University, 2Vanderbilt Center for Bone Biology, Vanderbilt University, 3Department of Veterans Affairs, Tennessee Valley Healthcare System (VISN 9), 4Department of Medicine, Division of Clinical Pharmacology, Vanderbilt University, 5Department of Cancer Biology, Vanderbilt University Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models. Medicine Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-κB Reporter Transgenic Whitney Barham1, Taylor Sherrill2, Linda Connelly3, Timothy S. Blackwell2, Fiona E. Yull1 1Cancer Biology Department, Vanderbilt University Medical Center, 2Department of Medicine, Vanderbilt University Medical Center, 3Department of Pharmaceutical Sciences, University of Hawaii at Hilo College of Pharmacy Described here is a technique in which lipopolysaccharide is injected into the lactating mouse mammary gland via the nipple to simulate mastitis, a condition commonly caused by bacterial infection. Lipopolysaccharide injection results in increased nuclear factor kappa B (NF-κB) signaling, visualized through bioluminescent imaging of an NF-κB luciferase reporter mouse. Neuroscience An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice Dennis P. Buehler1, Carrie B. Wiese1, S. B. Skelton1, E. Michelle Southard-Smith1 1Division of Genetic Medicine, Department of Medicine, Vanderbilt University School of Medicine An optimized procedure to purify neural crest-derived neuronal progenitors from fetal mouse tissues is described. This method takes advantage of expression from fluorescent reporter alleles to isolate discrete populations by fluorescence-activated cell sorting (FACS). The technique can be applied to isolate neuronal subpopulations throughout development or from adult tissues. Bioengineering High Throughput Single-cell and Multiple-cell Micro-encapsulation Todd P. Lagus1, Jon F. Edd1 1Department of Mechanical Engineering, Vanderbilt University Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops. Neuroscience Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube Elise R. Pfaltzgraff1, Nathan A. Mundell1,2, Patricia A. Labosky3 1Department of Cell and Developmental Biology, Center for Stem Cell Biology, Vanderbilt University Medical Center, 2Department of Pharmacology, Center for Stem Cell Biology, Vanderbilt University Medical Center, 3Vanderbilt University Medical Center Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest. Medicine The Polyvinyl Alcohol Sponge Model Implantation Desirae L. Deskins1,2, Shidrokh Ardestani1,2, Pampee P. Young1,2,3 1Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, 2The Department of Veterans Affairs Medical Center, 3Internal Medicine, Vanderbilt University School of Medicine A useful tool to analyze the effects of drugs, growth factors, and/or manipulated cells in an animal model of wound repair is described. This technique utilizes the properties of a polyvinyl alcohol (PVA) sponge to deliver and contain the desired treatment and also provide a platform to be excised and analyzed. Medicine Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice Julio E. Ayala1, Deanna P. Bracy2,3, Carlo Malabanan3, Freyja D. James2,3, Tasneem Ansari3, Patrick T. Fueger4, Owen P. McGuinness2,3, David H. Wasserman2,3 1Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute at Lake Nona, 2Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 3Vanderbilt Mouse Metabolic Phenotyping Center, Vanderbilt University School of Medicine, 4Department of Pediatrics and Cellular and Integrative Physiology, Indiana University School of Medicine The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress. Immunology and Infection In vitro Uncoating of HIV-1 Cores Vaibhav B. Shah1, Christopher Aiken1 1Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro. Medicine Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer's Disease Erin Hussey1, Brandon Ally1 1Department of Neurology, Vanderbilt University The methodology for collecting high-density event-related potential data while patients with Alzheimer's disease perform a recognition memory task is reviewed. This protocol will include subject preparation, quality assurance, data acquisition, and data analysis. Immunology and Infection Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging Jenny M. Tam1, Carlos E. Castro2, Robert J. W. Heath3, Michael K. Mansour1, Michael L. Cardenas1, Ramnik J. Xavier3, Matthew J. Lang4, Jatin M. Vyas1 1Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, 2Department of Mechanical and Aerospace Engineering, The Ohio State University, 3Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, 4Dept. of Chemical and Biomolecular Engineering, Vanderbilt University A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells. Medicine Microvascular Decompression: Salient Surgical Principles and Technical Nuances Jonathan Forbes1, Calvin Cooper*2, Walter Jermakowicz*2, Joseph Neimat*1, Peter Konrad*1 1Department of Neurosurgery, Vanderbilt University Medical Center, 2Vanderbilt School of Medicine, Vanderbilt University Medical Center There are many available options for management of the patient with trigeminal neuralgia. Microvascular decompression, while the most invasive of all options, is also the most effective at achieving long term remission of symptoms. Video instruction on how to maximize efficacy and minimize complications with this procedure is described. Medicine Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging Choi-Fong Cho1, Amber Ablack2, Hon-Sing Leong2, Andries Zijlstra3, John Lewis2,4 1Department of Medical Biophysics, University of Western Ontario, 2London Regional Cancer Program, London Health Science Centre, 3Department of Pathology, Vanderbilt University, 4Translational Prostate Cancer Research Group, London Health Science Centre We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model. Medicine Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model Trenis D. Palmer1, John Lewis2, Andries Zijlstra1 1Department of Pathology, Vanderbilt University, 2Departments of Oncology, Surgery and Medical Biophysics, London Regional cancer program Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs. Biology Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran Joshua A. Clanton1, Ilya A. Shestopalov2, James K. Chen2, Joshua T. Gamse1 1Department of Biological Sciences, Vanderbilt University, 2Department of Chemical and Systems Biology, Stanford University This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein. Biology Modified Mouse Embryonic Stem Cell based Assay for Quantifying Cardiogenic Induction Efficiency Ada Ao1, Charles H. Williams1, Jijun Hao1, Charles C. Hong1,2,3,4 1Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University School of Medicine, 2Department of Pharmacology, Vanderbilt University School of Medicine, 3Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, 4Research Medicine, Veterans Administration TVHS We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/β-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages. Biology Large Scale Zebrafish-Based In vivo Small Molecule Screen Jijun Hao1, Charles H. Williams1, Morgan E. Webb1, Charles C. Hong1,2,3,4 1Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University School of Medicine, 2Department of Pharmacology, Vanderbilt University School of Medicine, 3Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, 4Research Medicine, Veterans Affairs TVHS, Vanderbilt University School of Medicine Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos. Immunology and Infection Enzyme-linked Immunospot Assay (ELISPOT): Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens Isfahan R. Chambers*1, Tiffany R. Cone*1, Kyra Oswald-Richter2, Wonder P. Drake1,2 1Department of Medicine, Vanderbilt University School of Medicine, 2Department of Microbiology and Immunology, Vanderbilt University School of Medicine Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay. Neuroscience Operant Sensation Seeking in the Mouse Christopher M. Olsen1, Danny G. Winder1 1Department of Molecular Physiology and Biophysics, Center for Molecular Neuroscience, Kennedy Center for Human Development, Vanderbilt University Medical Center In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food. Neuroscience Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice Xi Huang1, Anuraag Sarangi2, Tatiana Ketova1, Ying Litingtung1, Michael K. Cooper2, Chin Chiang1 1Department of Cell and Developmental Biology, Vanderbilt University, 2Department of Neurology, Vanderbilt University This protocol describes the isolation and dissociation of mouse medulloblastoma tissue, and subsequent allografting of the tumor cells into immunocompromised recipient mice in order to initiate secondary medulloblastoma. Neuroscience Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells Xi Huang1, Tatiana Ketova1, Ying LItingtung1, Chin Chiang1 1Department of Cell and Developmental Biology, Vanderbilt University This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity. Biology Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells Gert-Jan Kremers1, David Piston1 1Department of Molecular Physiology and Biophysics, Vanderbilt University This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa. Biology Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media Dmitry A. Markov1,2, Philip C. Samson1, David K. Schaffer1, Adit Dhummakupt1, John P. Wikswo1,2,3,4, Leslie M. Shor5,6 1Vanderbilt Institute for Integrative Biosystems Research and Education, Vanderbilt University, 2Department of Biomedical Engineering, Vanderbilt University, 3Department of Molecular Physiology and Biophysics, Vanderbilt University, 4Department of Physics and Astronomy, Vanderbilt University, 5Department of Chemical, Materials and Biomolecular Engineering, University of Connecticut, 6Center for Environmental Sciences and Engineering, University of Connecticut Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface. Biology Murine Colitis Modeling using Dextran Sulfate Sodium (DSS) Caitlyn G. Whittem1, Amanda D. Williams2, Christopher S. Williams2 1Department of Cancer Biology, Vanderbilt University, 2Departments of Medicine and Cancer Biology, Vanderbilt University Dextran sulfate sodium (DSS) administered in the drinking water is an established murine inflammatory injury model of acute colitis. This protocol outlines the method for DSS treatment and the preparation of tissues. Biology Electrophysiological Recording in the Drosophila Embryo Kaiyun Chen1, David E. Featherstone1, Kendal Broadie2 1Department of Biological Sciences, University of Illinois, 2Department of Biological Sciences, Vanderbilt University Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses. Biology Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures David E. Featherstone1, Kaiyun Chen1, Kendal Broadie2 1Department of Biological Sciences, University of Illinois, 2Department of Biological Sciences, Vanderbilt University This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.
