JoVE: Journal of Visualized Experiments

High-throughput Detection Method for Influenza Virus

Sat, 04 February 2012 14:00:00 EST

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires <10 μl volume of test material and has the potential for concurrent screening of multiple pathogens.

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Fri, 03 February 2012 13:00:00 EST

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Fri, 03 February 2012 12:00:00 EST

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

Thu, 02 February 2012 13:00:00 EST

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma

Thu, 02 February 2012 12:00:00 EST

Anodic arc discharge is one of the most practical and efficient methods to synthesize various carbon nanostructures. To increase the arc controllability and flexibility, a non-uniform magnetic field was introduced to process the one-step synthesis of large-scale graphene flakes and high-purity single-walled carbon nanotubes.

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Wed, 01 February 2012 13:00:00 EST

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition

Wed, 01 February 2012 12:00:00 EST

We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Tue, 31 January 2012 13:00:00 EST

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

Tue, 31 January 2012 12:00:00 EST

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Mon, 30 January 2012 13:00:00 EST

Stereotaxic surgery on rodents allows for targeted administration of drugs or electrical stimulation and recordings in awake, behaving animals. In this video presentation we will demonstrate recent procedural refinements to this long-standing procedure that successfully improved survival rate and reduced post-surgical weight loss.

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

Mon, 30 January 2012 12:00:00 EST

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.

The Mouse Forced Swim Test

Sun, 29 January 2012 14:00:00 EST

The forced swim test is validated as an experimental approach to assess potential antidepressant efficacy in rodents. Experimental animals are placed in a tank of water and escape-related mobility behavior is quantified. The common procedures for the mouse version of this test are described.

The Tail Suspension Test

Sat, 28 January 2012 21:00:00 EST

The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test.

Measuring Peptide Translocation into Large Unilamellar Vesicles

Fri, 27 January 2012 13:00:00 EST

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

Fri, 27 January 2012 12:00:00 EST

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Thu, 26 January 2012 13:00:00 EST

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Thu, 26 January 2012 12:00:00 EST

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Polymer Microarrays for High Throughput Discovery of Biomaterials

Wed, 25 January 2012 13:00:00 EST

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Quantifying Mixing using Magnetic Resonance Imaging

Wed, 25 January 2012 12:00:00 EST

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

Intracellular Refolding Assay

Tue, 24 January 2012 13:00:00 EST

In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

Tue, 24 January 2012 12:00:00 EST

Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

Pull-down of Calmodulin-binding Proteins

Mon, 23 January 2012 13:00:00 EST

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Mon, 23 January 2012 12:00:00 EST

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

The Use of Thermal Infra-Red Imaging to Detect Delayed Onset Muscle Soreness

Sun, 22 January 2012 14:00:00 EST

The purpose of this investigation was to assess whether using an infra-red thermal camera is a valid tool for detecting and quantifying the muscle soreness after exercising.

Analysis of Cell Cycle Position in Mammalian Cells

Sat, 21 January 2012 14:00:00 EST

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Real-time fMRI Biofeedback Targeting the Orbitofrontal Cortex for Contamination Anxiety

Fri, 20 January 2012 13:00:00 EST

Here we present a method for training people to control a brain area involved in contamination anxiety and for probing the relationship between contamination anxiety and brain connectivity patterns.

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Fri, 20 January 2012 12:00:00 EST

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Thu, 19 January 2012 13:00:00 EST

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

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

Thu, 19 January 2012 12:00:00 EST

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.

Assaying the Kinase Activity of LRRK2 in vitro

Wed, 18 January 2012 13:00:00 EST

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

Wed, 18 January 2012 12:00:00 EST

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Tue, 17 January 2012 13:00:00 EST

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

Tue, 17 January 2012 12:00:00 EST

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

Mon, 16 January 2012 13:00:00 EST

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Mon, 16 January 2012 12:00:00 EST

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

Sun, 15 January 2012 14:00:00 EST

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

Sat, 14 January 2012 14:00:00 EST

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

Fri, 13 January 2012 13:00:00 EST

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

Fri, 13 January 2012 12:00:00 EST

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Thu, 12 January 2012 13:00:00 EST

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

Thu, 12 January 2012 12:00:00 EST

An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Wed, 11 January 2012 13:00:00 EST

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

Wed, 11 January 2012 12:00:00 EST

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Tue, 10 January 2012 13:00:00 EST

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

Immunostaining of Dissected Zebrafish Embryonic Heart

Tue, 10 January 2012 12:00:00 EST

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Mon, 09 January 2012 13:00:00 EST

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Isolation and Characterization of RNA-Containing Exosomes

Mon, 09 January 2012 12:00:00 EST

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

Sun, 08 January 2012 14:00:00 EST

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Sat, 07 January 2012 14:00:00 EST

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of “niches” within the thymus where T cell development occurs.

High Content Screening in Neurodegenerative Diseases

Fri, 06 January 2012 13:00:00 EST

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.