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The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.

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 JoVE Neuroscience

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient


JoVE 3196 9/17/2011

1, 1, 2, 1

1Department of Pathology and Laboratory Medicine, Waisman Center for Developmental Disabilities, University of Wisconsin, 2Department of Biochemistry, Waisman Center for Developmental Disabilities, University of Wisconsin

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

 JoVE Immunology and Infection

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient


JoVE 1420 11/03/2009

1, 2, 3, 2,4

1Dynamac, Inc., 2Department of Biological Sciences, University of Cincinnati, McMicken College of Arts and Science, 3Animal Parasitic Disease Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 4National Exposure Research Laboratory, US Environmental Protection Agency

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

 JoVE Clinical and Translational Medicine

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer


JoVE 4321 10/10/2012

1,2, 1,2,3, 1,2,3

1Department of Medicine, Baylor College of Medicine, 2Division of Diabetes, Endocrinology & Metabolism, Diabetes & Endocrinology Research Center, Baylor College of Medicine, 3Department of Molecular & Cellular Biology, Baylor College of Medicine

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

 JoVE Clinical and Translational Medicine

Isolation of Human Islets from Partially Pancreatectomized Patients


JoVE 2962 7/30/2011

1, 1, 1, 2, 1, 1, 3, 2, 1

1Department of GI-, Thorax- and Vascular Surgery, University Hospital Carl Gustav Carus, University of Technology Dresden, 2Molecular Diabetology, Paul Langerhans Institute Dresden, 3Department of Pathology, University Hospital Carl Gustav Carus, University of Technology Dresden

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

 JoVE General

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons


JoVE 990 1/16/2009

1, 2, 2,3, 2,4

1Department of Genetics and Development, Columbia University, 2Department of Pathology and Cell Biology, Columbia University, 3Department of Neuroscience, Columbia University, 4Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

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 JoVE General

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides


JoVE 4027 6/19/2012

,

Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

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 JoVE Clinical and Translational Medicine

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease


JoVE 4338 11/28/2012

1, 2, 1

1Department of Surgery, The University of Alabama at Birmingham, 2Department of Medicine, The University of Alabama at Birmingham

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

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 JoVE Bioengineering

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters


JoVE 2494 1/21/2011

1, 1,2, 3, 1,3,4

1Department of Biomedical Engineering, University of Michigan, 2State Key Laboratory of Bioelectronics, Southeast University, 3Department of Neurology, University of Michigan, 4Geriatrics Research, Education and Clinical Center, Veterans Affairs Ann Arbor Healthcare Center

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

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 JoVE Immunology and Infection

In vitro Uncoating of HIV-1 Cores


JoVE 3384 11/08/2011

,

Department 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.

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 JoVE Chemistry

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)


JoVE 50242 3/14/2013

, ,

Proteomics and Metabolomics Facility, Colorado State University

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

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 JoVE Clinical and Translational Medicine

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors


JoVE 2538 3/22/2011

, , , , , , , ,

Department of Pediatrics, Division of Cellular and Molecular Therapy, University of Florida

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

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 JoVE General

Identification of Protein Interacting Partners Using Tandem Affinity Purification


JoVE 3643 2/25/2012

*, *, ,

Section of Virology, Department of Medicine, Imperial College London

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

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 JoVE Bioengineering

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy


JoVE 50310 4/04/2013

1, 1, 2, 1, 1, 2, 1, 1

1Centre for Vascular Research and Australian Centre for Nanomedicine, The University of New South Wales, 2School of Chemistry and Australian Centre for Nanomedicine, The University of New South Wales

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

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 JoVE General

A Gradient-generating Microfluidic Device for Cell Biology


JoVE 271 8/30/2007

, , , , ,

Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology; Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

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 JoVE General

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients


JoVE 1779 4/19/2010

1, 2, 1,3

1McFerrin Department of Chemical Engineering, Texas A&M University, 2Department of Biology, Texas A&M University, 3Department of Biomedical Engineering, Texas A&M University

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

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 JoVE Bioengineering

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation


JoVE 3855 1/22/2013

1,2,3, 4, 1,2, 1,2, 2,3, 1,2,3, 2,3,5

1Department of Pathology, Harvard Medical School, 2Massachusetts General Hospital, 3Wellman Center for Photomedicine, Harvard Medical School, 4Pulmonary and Critical Care Unit, Massachusetts General Hospital, 5Pulmonary and Critical Care Unit, Harvard Medical School

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

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 JoVE General

DNA Stable-Isotope Probing (DNA-SIP)


JoVE 2027 8/02/2010

,

Department of Biology, University of Waterloo

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

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 JoVE General

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI


JoVE 3362 1/30/2012

,

Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center, College of Medicine

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

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 JoVE Chemistry

Fabricating Nanogaps by Nanoskiving


JoVE 50406 5/13/2013

,

Stratingh Institute for Chemistry and Zernike Institute for Advanced Materials, University of Groningen

The fabrication of electrically addressable, high-aspect-ratio (> 1000:1) metal nanowires separated by gaps of single nanometers using either sacrificial layers of aluminum and silver or self-assembled monolayers as templates is described. These nanogap structures are fabricated without a clean room or any photo- or electron-beam lithographic processes by a form of edge lithography known as nanoskiving.

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 JoVE Neuroscience

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay


JoVE 3330 1/19/2012

1, 2, 1

1Department of Biology, California State University, Northridge, 2Centro de Biología Celular y Molecular, Universidad Nacional de Córdoba

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

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 JoVE Neuroscience

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain


JoVE 3746 5/10/2012

, ,

Psychology & Neurobiology, Imaging Research Center & Center for Perceptual Systems, The University of Texas at Austin

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

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 JoVE General

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae


JoVE 638 1/03/2008

, ,

Laboratory of Neurogenetics and Behavior, Rockefeller University

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

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 JoVE Immunology and Infection

Seven Steps to Stellate Cells


JoVE 2710 5/10/2011

, ,

Immune Disease Institute, Program in Cellular and Molecular Medicine at Children's Hospital, Department of Pathology, Harvard Medical School

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

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 JoVE General

Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders


JoVE 2897 9/04/2011

1, 1, 1,2

1Division of Endocrinology and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, 2Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine

Mutations in the kisspeptin receptor (KISS1R) are associated with reproductive disorders in patients. Here we describe how to introduce mutations of interest in the GC-rich sequence of KISS1R as well as the use of KISS1R constructs to characterize the degradation pathway of the receptor by immunoprecipitation and western blot.

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 JoVE General

Eukaryotic Polyribosome Profile Analysis


JoVE 1948 6/15/2010

, , , , , , ,

Department of Molecular Genetics, Microbiology, and Immunology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

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 JoVE General

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum


JoVE 3128 9/20/2011

,

Chemotaxis Signal Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

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 JoVE Clinical and Translational Medicine

Method to Measure Tone of Axial and Proximal Muscle


JoVE 3677 12/14/2011

1, 2, 1, 3

1Department of Biomedical Engineering, Oregon Health and Science University, 2UCL Institute of Neurology, Queen Square, 3Department of Neurology, Oregon Health and Science University

We have developed a device (Twister) to study the regulation of tonic muscle activity during active postural maintenance. Twister measures torsional resistance and muscular responses in standing subjects during twisting of the body axis. The device can be flexibly configured to study various aspects of tonic control across the neck, trunk, and/or hips.

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 JoVE General

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets


JoVE 1343 5/26/2009

, , , , ,

Department of Surgery, University of Illinois, Chicago

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

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 JoVE General

Two Types of Assays for Detecting Frog Sperm Chemoattraction


JoVE 3407 12/27/2011

1, 2, 2

1Department of Animal Sciences, University of Illinois, Urbana-Champaign, 2School of Life Sciences, Arizona State University

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction – sperm accumulation assays and sperm tracking assays.

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 JoVE Clinical and Translational Medicine

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model


JoVE 3996 8/04/2012

1, 2,3

1Department of Applied Physics and Physico-Informatics, Keio University, 2Department of Biochemistry, School of Medicine, Keio University, 3Exploratory Research for Advanced Technology (ERATO), Suematsu Gas Biology Project, Japan Science and Technology Agency (JST)

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

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 JoVE General

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli


JoVE 2505 3/16/2011

, , , ,

Department of Microbiology & Environmental Toxicology, University of California Santa Cruz - UCSC

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

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 JoVE Immunology and Infection

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry: a Novel Use for a Cell Purification Method


JoVE 2698 4/09/2011

1,2,3,4, 5, 1,2,3,4,6

1Institute for Memory Impairments and Neurological Disorders, University of California, 2Physical Medicine & Rehabilitation, University of California, 3Anatomy & Neurobiology, University of California, 4Sue and Bill Gross Stem Cell Research Center, University of California, 5Section of Molecular Biology, University of California, 6Reeve-Irvine Research Center, University of California

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

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 JoVE Chemistry

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies


JoVE 50141 3/06/2013

1,2, 1, 1,2, 1,2, 1,2, 1,2

1Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, 2The University of Melbourne

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

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 JoVE Immunology and Infection

Isolation of Lymphocytes from Mouse Genital Tract Mucosa


JoVE 4391 9/03/2012

1, 1,2

1Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, 2California NanoSystems

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

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 JoVE General

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level


JoVE 1484 9/07/2009

1, 1,2, 1

1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 2Howard Hughes Medical Institute, Harvard Medical School

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

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