JoVE Logo

Sign In

Vanderbilt University School of Medicine

28 ARTICLES PUBLISHED IN JoVE

image

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-Richter 2, Wonder P. Drake 1,2
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.

image

Biology

Large Scale Zebrafish-Based In vivo Small Molecule Screen
Jijun Hao 1, Charles H. Williams 1, Morgan E. Webb 1, Charles C. Hong 1,2,3,4
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.

image

Biology

Modified Mouse Embryonic Stem Cell based Assay for Quantifying Cardiogenic Induction Efficiency
Ada Ao 1, Charles H. Williams 1, Jijun Hao 1, Charles C. Hong 1,2,3,4
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.

image

Medicine

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice
Julio E. Ayala 1, Deanna P. Bracy 2,3, Carlo Malabanan 3, Freyja D. James 2,3, Tasneem Ansari 3, Patrick T. Fueger 4, Owen P. McGuinness 2,3, David H. Wasserman 2,3
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.

image

Immunology and Infection

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure
Danielle L. Michell 1, Karen L. Andrews 1, Kevin J. Woollard 1, Jaye P.F. Chin-Dusting 1
1Vascular Pharmacology Laboratory, Baker IDI Heart and Diabetes Institute, Monash University

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

image

Immunology and Infection

In vitro Uncoating of HIV-1 Cores
Vaibhav B. Shah 1, Christopher Aiken 1
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.

image

Medicine

The Polyvinyl Alcohol Sponge Model Implantation
Desirae L. Deskins 1,2, Shidrokh Ardestani 1,2, Pampee P. Young 1,2,3
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.

image

Immunology and Infection

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models
Wei Han 1, Hui Li 1, Brahm H. Segal 2,3, Timothy S. Blackwell 1
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.

image

Medicine

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-κB Reporter Transgenic
Whitney Barham 1, Taylor Sherrill 2, Linda Connelly 3, Timothy S. Blackwell 2, Fiona E. Yull 1
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.

image

Neuroscience

An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice
Dennis P. Buehler 1, Carrie B. Wiese 1, S. B. Skelton 1, E. Michelle Southard-Smith 1
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.

image

Biology

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels
Rene Raphemot 1,2, C. David Weaver 1,3, Jerod S. Denton 1,2
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.

image

Medicine

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics
Jacob A. Martin 1, Alexander S. Maris 1, Moneeb Ehtesham 1, Robert J. Singer 1
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.

image

Immunology and Infection

Isolation of Adipose Tissue Immune Cells
Jeb S. Orr 1, Arion J. Kennedy 1, Alyssa H. Hasty 1
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.

image

JoVE Journal

Reconstitution Of β-catenin Degradation In Xenopus Egg Extract
Tony W. Chen *1, Matthew R. Broadus *1, Stacey S. Huppert 2, Ethan Lee 1,3
1Department of Cell and Developmental Biology and Program in Developmental Biology, Vanderbilt University Medical Center, 2Division of Gastroenterology, Hepatology & Nutrition and Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 3Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

image

Medicine

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens
Kasey Jividen *1, Mercedeh Javanbakht Movassagh *1, Amir Jazaeri 2, Hui Li 1
1Department of Pathology, University of Virginia, 2Department of Obstetrics & Gynecology, University of Virginia

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients. 

image

Immunology and Infection

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability
Kathryn Patricia Haley 1, Eric Joshua Blanz 1, Jennifer Angeline Gaddy 1,2
1Department of Medicine - Division of Infectious Diseases, Vanderbilt University School of Medicine, 2Tennessee Valley Healthcare Systems, U. S. Dept. of Veterans Affairs

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

image

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.

image

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.

image

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.

image

Biochemistry

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification
Danielle L. Michell *1, Ryan M. Allen *1, Stuart R. Landstreet 1, Shilin Zhao 2, Cynthia L. Toth 1, Quanhu Sheng 3, Kasey C. Vickers 1,2
1Department of Medicine, Vanderbilt University School of Medicine, 2Center for Quantitative Sciences, Vanderbilt University School of Medicine, 3Department of Cancer Biology, Vanderbilt University School of Medicine

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

image

JoVE Core

Preparation of Aligned Steel Fiber Reinforced Cementitious Composite and Its Flexural Behavior
Ru Mu 1, Luansu Wei 1, Xiaowei Wang 1, Hui Li 1, Longbang Qing 1, Jian Zhou 1, Quanming Zhao 2
1School of Civil and Transportation Engineering, Hebei University of Technology, 2School of Information Engineering, Hebei University of Technology

This protocol describes an approach for manufacturing aligned steel fiber reinforced cementitious composite by applying a uniform electromagnetic field. Aligned steel fiber reinforced cementitious composite exhibits superior mechanical properties to ordinary fiber reinforced concrete.

image

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.

image

Neuroscience

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue
Aaron A. May-Zhang 1, Karen K Deal 1, E. Michelle Southard-Smith 1
1Vanderbilt University Medical Center

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

image

Cancer Research

Perfusion and Inflation of the Mouse Lung for Tumor Histology
Mackenzie L. Davenport 1, Taylor P. Sherrill 2, Timothy S. Blackwell 2, Mick D. Edmonds 1
1Department of Genetics, University of Alabama at Birmingham, 2Department of Medicine, Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University School of Medicine

The purpose of this method is to present a simple and efficient method for the perfusion, inflation, and fixation of mouse lungs for the examination of lung tumor pathology and evaluation of metastases to the lung.

image

Behavior

Using a Virtual Reality Walking Simulator to Investigate Pedestrian Behavior
Hyun Chae Chung 1, Soon Ho Kim 2, Gyoojae Choi 3, Jong Won Kim 4, Moo Young Choi 2, Hui Li 1
1Department of Sport and Exercise Sciences, Kunsan National University, 2Department of Physics and Astronomy, Seoul National University, 3School of Mechanical and Automotive Engineering, Kunsan National University, 4Department of Healthcare Information Technology, Inje University

This protocol describes use of a walking simulator that serves as a safe and ecologically valid method to study pedestrian behavior in the presence of moving traffic.

image

Medicine

Blind Endotracheal Intubation in Neonatal Rabbits
Sergio Benito 1, Stephanie Hadley 2, Marta Camprubí-Camprubí 3, Joan Sanchez-de-Toledo 4,5
1Department of Pediatric Critical Care, Hospital Sant Joan de Déu, Institut de Recerca Sant Joan de Déu, 2Vanderbilt University School of Medicine, 3Neonatology Department, Hospital Sant Joan de Déu, Institut de Recerca Sant Joan de Déu, Universidad de Barcelona, 4Department of Pediatric Cardiology, Hospital Sant Joan de Déu, Institut de Recerca Sant Joan de Déu, 5Department of Critical Care Medicine, University of Pittsburgh

We describe a technique of endotracheal intubation in newborn rabbits after esophageal catheterization with a gastric tube.

image

Biology

Optimization of Transesophageal Atrial Pacing to Assess Atrial Fibrillation Susceptibility in Mice
Matthew B. Murphy 1, Kyungsoo Kim 1, Prince J. Kannankeril 1, Katherine T. Murray 1
1Departments of Medicine, Pediatrics and Pharmacology, Vanderbilt University School of Medicine

The present protocol describes the optimization of experimental parameters when using transesophageal atrial pacing to assess atrial fibrillation susceptibility in mice.

image

Biology

Human Pseudoislet System for Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Secretory Profiles
Tiffany M. Richardson *1, Yasminye D. Pettway *1, John T. Walker 1, Heather A. Nelson 2, Matthew Ishahak 3, Gregory Poffenberger 2, Radhika Aramandla 2, Conrad Reihsmann 2, Ashutosh Agarwal 3, Alvin C. Powers 1,2,4, Marcela Brissova 2
1Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, 3Department of Biomedical Engineering, University of Miami, 4VA Tennessee Valley Healthcare System

This protocol describes a method for the synchronous acquisition and co-registration of intracellular signaling events and the secretion of insulin and glucagon by primary human pseudoislets using the adenoviral delivery of a cyclic adenosine monophosphate (cAMP) biosensor, a cAMP difference detector in situ (cADDis), and a microperifusion system.

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved