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67 ARTICLES PUBLISHED IN JoVE

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Biology

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures
David E. Featherstone 1, Kaiyun Chen 1, Kendal Broadie 2
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.

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Biology

Electrophysiological Recording in the Drosophila Embryo
Kaiyun Chen 1, David E. Featherstone 1, Kendal Broadie 2
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.

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Biology

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)
Caitlyn G. Whittem 1, Amanda D. Williams 2, Christopher S. Williams 2
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.

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Biology

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media
Dmitry A. Markov 1,2, Philip C. Samson 1, David K. Schaffer 1, Adit Dhummakupt 1, John P. Wikswo 1,2,3,4, Leslie M. Shor 5,6
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.

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Biology

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells
Gert-Jan Kremers 1, David Piston 1
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.

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Neuroscience

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells
Xi Huang 1, Tatiana Ketova 1, Ying LItingtung 1, Chin Chiang 1
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.

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Neuroscience

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice
Xi Huang 1, Anuraag Sarangi 2, Tatiana Ketova 1, Ying Litingtung 1, Michael K. Cooper 2, Chin Chiang 1
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.

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Biology

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran
Joshua A. Clanton 1, Ilya A. Shestopalov 2, James K. Chen 2, Joshua T. Gamse 1
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.

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Medicine

Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer's Disease
Erin Hussey 1, Brandon Ally 1
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.

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Immunology and Infection

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging
Jenny M. Tam 1, Carlos E. Castro 2, Robert J. W. Heath 3, Michael K. Mansour 1, Michael L. Cardenas 1, Ramnik J. Xavier 3, Matthew J. Lang 4, Jatin M. Vyas 1
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.

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Bioengineering

High Throughput Single-cell and Multiple-cell Micro-encapsulation
Todd P. Lagus 1, Jon F. Edd 1
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.

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Neuroscience

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube
Elise R. Pfaltzgraff 1, Nathan A. Mundell 1,2, Patricia A. Labosky 3
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.

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Medicine

Models of Bone Metastasis
J. Preston Campbell 1,2, Alyssa R. Merkel 2,3,4, S. Kathryn Masood-Campbell 2,4, Florent Elefteriou 1,2,4,5, Julie A. Sterling 2,3,4,5
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.

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Medicine

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System
Teagan J. Walter 1,2, Erin E. Sparks 3, Stacey S. Huppert 2
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.

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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. Beavers 2,3, Arnold J. Kim 1, Hongmei Li 1,2, Heather M. Nelson 4, Todd D. Giorgio 1,2,5,6, Craig L. Duvall 1,2
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.

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Medicine

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation
J. Geraldo Valadez 1, Anuraag Sarangi 1, Christopher J. Lundberg 1, Michael K. Cooper 1,2,3
1Department of Neurology, Vanderbilt University Medical Center, 2Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, 3Neurology Service, Veteran Affairs TVHS

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models.

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Behavior

Quantitative Assessment of Cortical Auditory-tactile Processing in Children with Disabilities
Nathalie L. Maitre 1, Alexandra P. Key 2,3
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.

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Biology

Isolation and Physiological Analysis of Mouse Cardiomyocytes
Gretchen M. Roth 1, David M. Bader 1,2, Elise R. Pfaltzgraff 2
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.

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Medicine

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease
Bruce M. Damon 1,2,3,4, Ke Li 1,2, Richard D. Dortch 1,2, E. Brian Welch 1,2, Jane H. Park 1,2,4, Amanda K. W. Buck 1,2, Theodore F. Towse 1,2,5, Mark D. Does 1,2,3, Daniel F. Gochberg 1,2,6, Nathan D. Bryant 1,2
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.

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Medicine

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images
Aliya Gifford 1, Theodore F. Towse 2, Ronald C. Walker 3, Malcolm J. Avison 4, E. Brian Welch 3
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.

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Neuroscience

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons
Melanie Martin 1,2, Ke Li 1, Megan C. Wright 2, Angelo C. Lepore 1
1Department of Neuroscience, Farber Institute for Neurosciences, Sidney Kimmel Medical College at Thomas Jefferson University, 2Department of Biology, Arcadia University

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

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Behavior

Testing Sensory and Multisensory Function in Children with Autism Spectrum Disorder
Sarah H. Baum 1, Ryan A. Stevenson 2, Mark T. Wallace 3
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.

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Chemistry

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II
Keersten M. Davis 1, Anna L. Bitting 1, Christine F. Markwalter 1, Westley S. Bauer 1, David W. Wright 1
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.

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Neuroscience

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals
Feng Liu 1, Nannan Liu 1
1Department of Entomology and Plant Pathology, Auburn University

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

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Engineering

Data Acquisition Protocol for Determining Embedded Sensitivity Functions
Janette J. Meyer 1, Douglas E. Adams 1, Janene Silvers 2
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.

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JoVE Core

Printing Fabrication of Bulk Heterojunction Solar Cells and In Situ Morphology Characterization
Feng Liu 1, Sunzida Ferdous 2, Xianjian Wan 1, Chenhui Zhu 3, Eric Schaible 3, Alexander Hexemer 3, Cheng Wang 3, Thomas P. Russell 1,2
1Materials Sciences Division, Lawrence Berkeley National Laboratory, 2Department of Polymer Science and Engineering, University of Massachusetts, Amherst, 3Advanced Light Source, Lawrence Berkeley National Laboratory

Here, we present a protocol to fabricate organic thin film solar cells using a mini-slot die coater and related in-line structure characterizations using synchrotron scattering techniques.

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Developmental Biology

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2
Jeffery B. Bylund 1,2, Antonis K. Hatzopoulos 1,3
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.

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Bioengineering

Sustained Administration of β-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres
Raymond C. Pasek 1, Taylor E. Kavanaugh 2, Craig L. Duvall 2, Maureen A. Gannon 1,3
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.

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JoVE Journal

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision
Andrea A. Perreault 1, Bryan J. Venters 1
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.

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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 Laroumanie 1, Bethany L. Dale 2, Mohamed A. Saleh 3, Meena S. Madhur 1
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.

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Biochemistry

Expression, Purification, and Antimicrobial Activity of S100A12
Emmanuel Jackson 1, Saffron Little 1, Dana S. Franklin 1, Jennifer A. Gaddy 2,3, Steven M. Damo 1,4
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.

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JoVE Journal

Nanosponge Tunability in Size and Crosslinking Density
Laken L. Kendrick-Williams 1, Eva Harth 1
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.

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Genetics

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing
Ahmed Elaswad *1,2, Karim Khalil *1,3, David Cline 1, Patrick Page-McCaw 4, Wenbiao Chen 4, Maximilian Michel 5, Roger Cone 5, Rex Dunham 1
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.

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Developmental Biology

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis
Junhua Lv 1,2, Feng Liu 1,2
1State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

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Chemistry

One-pot Microwave-assisted Conversion of Anomeric Nitrate-esters to Trichloroacetimidates
D. Jamin Keith 1, Stefan A. Marasligiller *1, Alexander W. Sasse *1, Steven D. Townsend 1,2
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.

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Developmental Biology

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice
David E. Maridas 1, Elizabeth Rendina-Ruedy 1, Phuong T. Le 1, Clifford J. Rosen 1
1Maine Medical Center Research Institute

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

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Medicine

Fat-Water Phantoms for Magnetic Resonance Imaging Validation: A Flexible and Scalable Protocol
Emily C. Bush 1, Aliya Gifford 2, Crystal L. Coolbaugh 1, Theodore F. Towse 1,3,4, Bruce M. Damon 1,5,6,7, E. Brian Welch 1,5
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.

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JoVE Journal

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation
Danielle L. Kopke 1, Kendal Broadie 2
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.

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Genetics

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions
Stephanie S.L. Birnbaum 1, David C. Rinker 1, Patrick Abbot 1
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.

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Biochemistry

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation
Anne M. Scott 1, Ke Li 1, Weiming Li 1
1Department of Fisheries and Wildlife, Michigan State University

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

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Immunology and Infection

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells
Justin P. Van Beusecum 1, Liang Xiao 1, Natalia R. Barbaro 1, David M. Patrick 1, Annet Kirabo 1
1Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into naïve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

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Engineering

Evaluating Targeting Accuracy in the Focal Plane for an Ultrasound-guided High-intensity Focused Ultrasound Phased-array System
Ke Li 1,2, Jingfeng Bai 1,2, Yazhu Chen 1,2, Xiang Ji 1,2
1Biomedical Instrument Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, 2Shanghai Med-X Engineering Center for Medical Equipment and Technology, Shanghai Jiao Tong University

This study describes a protocol to evaluate the targeting accuracy in the focal plane of an ultrasound-guided high-intensity focused ultrasound phased-array system.

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Bioengineering

Growth and Characterization of Irradiated Organoids from Mammary Glands
Benjamin C. Hacker 1, Javier D. Gomez 1, Carlos A. Silvera Batista 1, Marjan Rafat 1,2,3
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.

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Cancer Research

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels
Steven M Alves 1, Tian Zhu 1, Anastasia Shostak 1, Ninna S. Rossen 2, Marjan Rafat 1,3,4
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.

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Developmental Biology

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence
Zhentao Zhang 1,2,3, Young-Jae Nam 1,2,3
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.

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Developmental Biology

Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption
Joseph M. Elsakr 1, Charles Deeter 2, Valerie Ricciardi 3, Maureen Gannon 1,4,5,6
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.

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Chemistry

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)
Albert B. Arul 1, Renã A.S. Robinson 1,2,3,4
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.

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Bioengineering

Low-Cost, Volume-Controlled Dipstick Urinalysis for Home-Testing
Emily Kight 1, Iftak Hussain 1, Audrey K. Bowden 1,2
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.

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Medicine

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development
Brian A. Juber 1, Timothy G. Elgin 1, Erin M. Fricke 2, Huyiu Gong 1, Jeffrey Reese 3, Steven J. McElroy 1,4
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.

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Neuroscience

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes
Xin Wang *1, Zhou Chen *1,2, Yifan Wang 1, Feng Liu 1,3, Shanshan Jiang 1, Nannan Liu 1
1Department of Entomology and Plant Pathology, Auburn University, 2Departments of Biochemistry & Biophysics, and Cellular & Molecular Pharmacology, University of California, San Francisco, 3Department of Biological Sciences, Vanderbilt University

A protocol is presented that functionally characterizes mosquito ORs in response to human odors using a Xenopus oocyte expression system coupled with a two-electrode voltage clamp, providing a powerful new technique for exploring the responses of mosquitoes ORs to exposure to human odors.

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Developmental Biology

Assessing Cardiac Reprogramming using High Content Imaging Analysis
Zhentao Zhang 1,2,3, Young-Jae Nam 1,2,3
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 a protocol to quantify directly reprogrammed induced cardiomyocyte-like cells (iCMs) in vitro using high content imaging analysis. This method allows us to quantify the efficiency of cardiac reprogramming in an automated manner and to directly visualize iCMs.

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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 Pasic 3, Ashley Pitzer 4,6, Sean S. Davies 4, Justin H. Layer 5, Raymond L. Mernaugh 3, Annet Kirabo 4,6
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.

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Neuroscience

Imaging Dendritic Spines in Caenorhabditis elegans
Andrea Cuentas-Condori 1, D. M. Miller III 1,2
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.

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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 Ukita 1, John W. Stokes 1, W. Kelly Wu 1, Jennifer Talackine 1, Nancy Cardwell 1, Yatrik Patel 1, Clayne Benson 5, Caitlin T. Demarest 1, Erika B. Rosenzweig 3, Keith Cook 2, Emily J. Tsai 4, Matthew Bacchetta 1,6
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.

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Biology

Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro
Zhu Li 1, Matthew J. W. Hampton 1, Matthew B. Barajas 1, Matthias L. Riess 1,2,3
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.

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Biology

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics
Shobana Jayapalan 1,2, Ananya Nandy 1,2, Elizabeth Rendina-Ruedy 1,2,3
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.

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Medicine

A Heterotopic Mouse Model for Studying Laryngeal Transplantation
Maeve M. Kennedy *1, Egehan Salepci *1,2, Cheryl Myers 1, Marshall Strome 3,4, David G. Lott 1,5
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.

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Biology

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum
James S. White 1,2, Kimberly S. LaFever 2, Andrea Page-McCaw 1,2
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.

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Immunology and Infection

Murine Fecal Isolation and Microbiota Transplantation
Jeanne A. Ishimwe 1, Jianyong Zhong 2,3, Valentina Kon 2, Annet Kirabo 1
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.

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Methods For Studying Osteoenergetics And Metabolism
Elizabeth Rendina-Ruedy 1, Courtney M. Karner 2
1Department of Medicine, Clinical Pharmacology, Vanderbilt University Medical Center, 2Department of Internal Medicine, Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center

Methods For Studying Osteoenergetics And Metabolism

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Biology

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model
William J. Cleveland *1, Josephine E. Hees *1,2, Claudius Balzer 1,2, Hunter F. Douglas 1, Troy Stevens 3,4,5, Matthias L. Riess 1,6,7
1Anesthesiology, Vanderbilt University Medical Center, 2Anesthesiology, University of Greifswald, 3Department of Physiology and Cell Biology, University of South Alabama, 4Department of Medicine, University of South Alabama, 5Center for Lung Biology, University of South Alabama, 6Anesthesiology, Tennessee Valley Healthcare System Veterans Affairs Medical Center, 7Pharmacology, Vanderbilt University

Ex vivo lungs are useful for a variety of experiments to collect physiological data while excluding the confounding variables of in vivo experiments. Commercial setups are often expensive and limited in the types of data they can collect. We describe a method for building a fully modular setup, adaptable for various study designs.

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Current and Novel Experimental Methods in Ischemia/Reperfusion Research: From Cutting-Edge Models to Innovative Therapies
Matthew B. Barajas 1,2, Matthias L. Riess 1,2,3
1Department of Anesthesiology, Vanderbilt University Medical Center, 2Department of Anesthesiology, TVHS VA Medical Center, 3Department of Pharmacology, Vanderbilt University

Current and Novel Experimental Methods in Ischemia/Reperfusion Research: From Cutting-Edge Models to Innovative Therapies

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Biology

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes
Adeline E. Williams 1,2, Irma Sanchez-Vargas 1, Lindsay E. Martin 2,3, Ines Martin-Martin 2,4, Susi Bennett 1, Ken E. Olson 1, Eric Calvo 2
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.

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Medicine

Pediatric Animal Model of Extracorporeal Cardiopulmonary Resuscitation After Prolonged Circulatory Arrest
Madeleine Ball *1,2, Sergio Benito *1,3, Juliana Pilar Caride 1, Cristina Ruiz-Herguido 1, Marta Camprubí-Camprubí 1,4, Joan Sanchez-de-Toledo 1,5,6
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.

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Medicine

A Porcine Model of Acute Respiratory Failure with a Continuous Infusion of Oleic Acid
Bret D. Alvis 1,2, Colleen Brophy 3, Joyce Cheung-Flynn 3, Marisa Case 1, Kyle Hocking 2,3
1Department of Anesthesiology, Vanderbilt University Medical Center, 2Department of Biomedical Engineering, Vanderbilt University, 3Department of Surgery, Vanderbilt University Medical Center

Infusing oleic acid continuously into the pulmonary artery of an anesthetized adult pig induces acute respiratory failure, enabling controlled experimentation during acute respiratory decompensation.

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Medicine

Adult and Pediatric Porcine Model of Acute Volume Overload
Bret D. Alvis 1,2, Jenna Helmer Sobey 1, Lexie Vaughn 3, Christina Jelly 1, Dawson Wervey 4, Joyce Cheung-Flynn 3, Marisa Case 1, Colleen Brophy 3, Kyle Hocking 2,3
1Department of Anesthesiology, Vanderbilt University Medical Center, 2Department of Biomedical Engineering, Vanderbilt University, 3Department of Surgery, Vanderbilt University Medical Center, 4Ohio University Heritage College of Osteopathic Medicine

The protocol here shows how continuous administration of crystalloids into the central veins of a euvolemic pig/piglet allows for the appropriate investigation of the physiological effects of acute volume overload.

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Preparation of Frozen Non-Human Primate Fetal Islets for Combined Single Nuclei RNA-Sequencing and ATAC-Sequencing, and Bulk Metabolomics
Darian T. Carroll 1, Sarah R. Lindsley 2, Melissa Kirigiti 2, Alex Buko 3, Angela Jones 4, Paul Kievit 2, Maureen Gannon 1,5,6,7
1Department of Molecular Physiology and Biophysics, Vanderbilt University, 2Division of Metabolic Health and Disease, Oregon National Primate Research Center, 3Human Metabolome Technologies (HMT) America, 4Vanderbilt Technologies for Advanced Genomics, Vanderbilt University Medical Center, 5Department of Medicine, Vanderbilt University Medical Center, 6Department of Veterans Affairs Tennessee Valley, 7Department of Cell and Developmental Biology, Vanderbilt University

This protocol describes procedures to isolate high-quality nuclei from frozen non-human primate pancreatic islets for use in single-nucleus simultaneous RNA sequencing and ATAC sequencing while preserving the bulk cytosolic fraction for metabolomic analyses from the same samples.

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