JoVE: Journal of Visualized Experiments

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Mon, 09 Nov 2009 17:00:00 EST

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

Digital Microfluidics for Automated Proteomic Processing

Fri, 06 Nov 2009 16:00:00 EST

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

Thu, 05 Nov 2009 16:00:00 EST

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Microvolume Protein Concentration Determination Using the NanoDrop Spectrophotometer

Wed, 04 Nov 2009 16:00:00 EST

Microvolume samples are quantified by a UV/Vis spectrophotometer that uses surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations without dilutions and speed by which they can be measured are greatly increased with this method.

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

Tue, 03 Nov 2009 16:00:00 EST

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

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Mon, 02 Nov 2009 16:05:00 EST

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

纳微量2000c 分光光度计- 微量体积蛋白浓度的测定

Sun, 01 Nov 2009 01:00:00 EST

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Fri, 30 Oct 2009 07:00:00 EST

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Thu, 29 Oct 2009 07:00:00 EST

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

Wed, 28 Oct 2009 07:00:00 EST

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.^1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Tue, 27 Oct 2009 07:00:00 EST

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

Mon, 26 Oct 2009 07:00:00 EST

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Fri, 23 Oct 2009 14:00:00 EST

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

Isolation and Derivation of Mouse Embryonic Germinal Cells

Thu, 22 Oct 2009 14:00:00 EST

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

Wed, 21 Oct 2009 14:00:00 EST

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Tue, 20 Oct 2009 14:00:00 EST

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

BioFlux System for Live Cell Experiments Under Controlled Shear Flow

Tue, 20 Oct 2009 00:00:00 EST

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

Mon, 19 Oct 2009 14:00:00 EST

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

Generation of Human CD40-activated B cells

Fri, 16 Oct 2009 23:30:00 EST

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

Thu, 15 Oct 2009 23:30:00 EST

Invertebrates show an autonomic sympathetic-like response similar to that described for vertebrates. The coordination of the cardio-vascular and ventilatory systems allows for measurement of a biological index in which to assess an organism internal state.

LAD-Ligation: A Murine Model of Myocardial Infarction

Wed, 14 Oct 2009 23:30:00 EST

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

Fluorescent Labeling of Drosophila Heart Structures

Tue, 13 Oct 2009 23:30:00 EST

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Mon, 12 Oct 2009 23:00:00 EST

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

Fri, 09 Oct 2009 16:30:00 EST

This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

Thu, 08 Oct 2009 16:03:00 EST

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Wed, 07 Oct 2009 17:25:00 EST

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

Tue, 06 Oct 2009 16:30:00 EST

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

Anterior Cervical Discectomy and Fusion in the Ovine Model

Mon, 05 Oct 2009 16:30:00 EST

This video demonstrates the technique of anterior cervical discectomy and fusion in the ovine model.

Lentivirus Production

Fri, 02 Oct 2009 21:00:00 EST

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Thu, 01 Oct 2009 21:00:00 EST

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

Wed, 30 Sep 2009 23:59:00 EST

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

Preparation and Using Phantom Lesions to Practice Fine Needle Aspiration Biopsies

Tue, 29 Sep 2009 21:00:00 EST

Practicing of fine needle aspiration biopsies (FNAB) by trainees is relatively challenging, due to the lack of an easily available, appropriate lesion. Preparation of an AV phantom lesion for practicing the FNAB procedure and mastering proficiency is relatively easy.

Visualizing the Beating Heart in Drosophila

Mon, 28 Sep 2009 21:00:00 EST

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

Microsurgical Clip Obliteration of Middle Cerebral Aneurysm Using Intraoperative Flow Assessment

Fri, 25 Sep 2009 15:10:00 EST

Description of the surgical obliteration of a cerebral aneurysm utilizing an ultrasonic flow probe to assess arterial flow prior to and after aneurysm clip placement.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Thu, 24 Sep 2009 15:00:00 EST

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

Large Insert Environmental Genomic Library Production

Wed, 23 Sep 2009 15:00:00 EST

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

Tue, 22 Sep 2009 15:00:00 EST

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Mon, 21 Sep 2009 15:00:00 EST

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

Biocision I - Sample Integrity at Any Temperature - Advertisement

Mon, 21 Sep 2009 01:00:00 EST

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Biocision II - Sample Integrity at Any Temperature - Advertisement

Mon, 21 Sep 2009 00:00:00 EST

DNA Extraction from 0.22 μM Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

Fri, 18 Sep 2009 05:00:00 EST

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 μm Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Thu, 17 Sep 2009 13:00:00 EST

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

Semi-automated Optical Heartbeat Analysis of Small Hearts

Wed, 16 Sep 2009 13:00:00 EST

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Tue, 15 Sep 2009 13:00:00 EST

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model

Mon, 14 Sep 2009 13:00:00 EST

This video demonstrates how to use a preclinical inexpensive and reliable model to study pathobiological and pathophysiological processes of in-stent restenosis development. Longitudinal in vivo monitoring using OCT (Optical Coherence Tomography) and analysis of OCT images are also demonstrated.

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Fri, 11 Sep 2009 16:30:00 EST

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

Thu, 10 Sep 2009 16:30:00 EST

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

Wed, 09 Sep 2009 16:30:00 EST

A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Tue, 08 Sep 2009 16:30:00 EST

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

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

Mon, 07 Sep 2009 16:00:00 EST

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.