JoVE Application Notes
Bio-Plex® Multi-Plex Immunoassay Overview and Testimonials - ADVERTISEMENT
Bio-Plex® overview with TGF-β mention
JoVE Clinical and Translational Medicine
Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds
Department of Anesthesia, Stanford University School of Medicine
This article provides a detailed and visual description of a methodology for collecting and measuring biochemical inflammatory and nociceptive mediators at the surgical wound site following cesarean delivery. This human bioassay has been used to determine correlations between wound and serum cytokine concentrations and drug-mediated changes in wound cytokines, chemokines and neuropetides.
JoVE General
Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System
1Department of Physical Electronics, Faculty of Engineering, Tel Aviv University, 2Department of Biomedical Engineering, Washington University in St. Louis, 3Department of Biological Sciences, University of Illinois, 4Department of Cell Research and Immunology, Tel Aviv University
Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.
JoVE General
Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds
Department of Anesthesiology, Stanford University School of Medicine
A technique to collect and measure surgical wound biochemical mediators at specific time points.
JoVE Immunology and Infection
Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres
1Saskatoon Research Centre, Agriculture and Agri-Food Canada, 2Department of Veterinary Microbiology, University of Saskatchewan, 3Plant Biotechnology Institute, National Research Council of Canada
We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.
JoVE Clinical and Translational Medicine
Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1β using a Whole Blood Stimulation Assay
1Translational Immunology Section, Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, 2Translational Autoinflammatory Disease Section, Office of the Clinical Director, National Institute of Arthritis and Musculoskeletal and Skin Diseases
We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.
JoVE Bioengineering
High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
1The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, 2Center for Reproductive Sciences, University of California San Francisco, 3Department of Urology, University of California San Francisco, 4Department of Cell and Tissue Biology, University of California San Francisco, 5Fluidigm Corporation, Fluidigm Corporation, 6Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Center, 7UCSF - Helen Diller Family Comprehensive Cancer Center, University of California San Francisco
Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.
JoVE General
Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms
1Medical Biochemistry and Molecular Biology, Saarland University, 2Experimental and Clinical Pharmacology and Toxicology, Saarland University
The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.
JoVE General
Scale-Up of Mammalian Cell Culture using a New Multilayered Flask
Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.
JoVE Neuroscience
Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits
Department of Biomedical Engineering, Washington University in St. Louis
We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.
JoVE Neuroscience
Forebrain Electrophysiological Recording in Larval Zebrafish
A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.
JoVE Bioengineering
Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study
1Department of Biomedical Engineering, Virginia Commonwealth University, 2Department of Anatomy and Neurobiology, Virginia Commonwealth University, 3Department of Cardiovascular Surgery, University Hospital of Geneva
The aim of this study was to mimic the native three layered architecture of the arterial wall. To accomplish this, electrospinning was employed with the use of a 3-1 (input-output) nozzle and blends of polycaprolactone, elastin, and collagen.
JoVE Clinical and Translational Medicine
Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion
Department of Neuroscience and Biomedical Technologies, University of Milano Bicocca
Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.
JoVE General
Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos
1Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, 2Departments of Neurology and Neurobiology, University of California, Los Angeles
Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.
JoVE General
An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase
Department of Biochemistry, University of Toronto
The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.
JoVE Neuroscience
Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound (fTCD)
Department of Experimental Psychology, University of Oxford
Functional transcranial Doppler sonography (fTCD) is a simple and non-invasive ultrasound technique which can be used to assess the lateralization of cognitive functions, especially language, and is suitable for use with children.
JoVE Applied Physics
Simulation, Fabrication and Characterization of THz Metamaterial Absorbers
School of Engineering, University of Glasgow
This protocol outlines the simulation, fabrication and characterization of THz metamaterial absorbers. Such absorbers, when coupled with an appropriate sensor, have applications in THz imaging and spectroscopy.
JoVE General
Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry
1Plant Molecular and Cellular Biology Program, University of Florida, 2Department of Biology, University of Florida, 3Interdisciplinary Center for Biotechnology Research, University of Florida, 4Genetics Institute, University of Florida
Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.
JoVE General
Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)
Centre for Infectious Diseases and Microbiology, University of Sydney
An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.
JoVE Neuroscience
Examining Local Network Processing using Multi-contact Laminar Electrode Recording
1Graduate School of Biomedical Science, Neuroscience Program, University of Texas, 2Department of Neurobiology and Anatomy, University of Texas
A fundamental issue in our understanding of cortical circuitry is how networks in different cortical layers encode sensory information. Here we describe electrophysiological techniques utilizing multi-contact laminar electrodes to record single-units and local field potentials and present analyses to identify cortical layers.
JoVE General
Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences
We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.
JoVE General
Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics
1Electrical Engineering Department, University of California, Los Angeles, 2California NanoSystems Institute, University of California, Los Angeles
Lensfree on-chip imaging and characterization of cells is illustrated. This on-chip cell imaging approach provides a compact and cost-effective tool for medical diagnostics and high-throughput cell biology applications, making it especially suitable for resource poor settings.
JoVE General
Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly
1Department of Brain and Cognitive Science, MIT - Massachusetts Institute of Technology, 2Picower Institute for Learning and Memory, MIT - Massachusetts Institute of Technology
In this protocol we demonstrate how to fabricate and condition tetrodes for use with a micro-drive array, which was designed for chronic electrophysiological recordings in rats. In addition, we illustrate the final stages of micro-drive array construction, which includes installing ground wires and a protective cone.
JoVE Application Notes
Profiling Changes in Receptor Tyrosine Kinase Phosphorylation using Antibody Arrays - ADVERTISEMENT
Array Group, Assay Department, R&D Systems, Inc.
Proteome Profiler antibody arrays are a convenient and cost efficient way to screen for changes in receptor tyrosine kinase (RTK) phosphorylation without performing numerous immunoprecipitation (IP) Westerns. The ARY001 Human RTK array allows for the qualitative measurement of multiple RTKs in a single sample using chemiluminescence detection.
JoVE Immunology and Infection
RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells
Department of Microbiology, Penn Genome Frontiers Institute, University of Pennsylvania
Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.
JoVE General
An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model
In this video, we demonstrate the EpiDerm Skin Irritation test (EpiDerm SIT) developed and validated for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients.
JoVE General
Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization
1Department of Cancer Genetics, BC Cancer Research Centre, 2Deeley Research Centre, BC Cancer Agency, 3Photography/Video Production, Multi-Media Services, BC Cancer Agency
This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.
JoVE General
The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project
1Applied Technology for Neuro-Psychology Lab, Istituto Auxologico Italiano, 2Psychology Department, Università Cattolica del Sacro Cuore
The Project on Virtual Reality Intelligent Multi-sensor System for the Treatment of Anxiety-related Disorders (INTREPID) is aimed at developing a multi-sensor context-aware virtual reality system for the treatment of anxiety-related disorders.
JoVE Application Notes
ヒト表皮再構築モデルを用いたIn vitro皮膚刺激試験 - ADVERTISEMENT
JoVE General
DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses
1Department of Integrative Oncology, BC Cancer Research Centre, 2Interdisciplinary Oncology Program, University of British Columbia - UBC, 3Photography/Video Production, Multi-Media Services, BC Cancer Agency, 4Department of Pathology and Laboratory Medicine, University of British Columbia - UBC
This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.
JoVE General
Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats
Animal Science Department, Iowa State University
Preimplantation embryos may be cryopreserved after placement into a hypertonic cryoprotective solution to cause cellular dehydration. After equilibration, ice crystal formation is induced in the solution surrounding the embryo. Further dehydration occurs as the embryo is slowly cooled to subzero temperatures before plunging into liquid nitrogen for storage.
JoVE Bioengineering
Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer
The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed
JoVE General
Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins
1Department of Radiation Oncology, Virginia Commonwealth University, 2Department of Biochemistry & Molecular Biology, Virginia Commonwealth University, 3Department of Anatomy & Neurobiology, Virginia Commonwealth University, 4Massey Cancer Center, Virginia Commonwealth University
This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.
JoVE Applied Physics
Determining 3D Flow Fields via Multi-camera Light Field Imaging
1Department of Mechanical Engineering, Brigham Young University, 2Naval Undersea Warfare Center, Newport, RI
A technique for performing quantitative three-dimensional (3D) imaging for a range of fluid flows is presented. Using concepts from the area of Light Field Imaging, we reconstruct 3D volumes from arrays of images. Our 3D results span a broad range including velocity fields and multi-phase bubble size distributions.
JoVE General
Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG
Macquarie Centre for Cognitive Science, Macquarie University
The advent of MEG systems sized for young children opens important new opportunities to study brain development. The new system, together with a protocol that aligns experimental requirements with the capacities of children, can be used to study cognitive and language processes in healthy, awake children aged three to six.
JoVE General
Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria
A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.
JoVE Editorial
March 2013: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here are some highlights from the March 2013 issue of Journal of Visualized Experiments (JoVE).
JoVE Immunology and Infection
Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus
1Center for Autophagy Research, Department of Internal Medicine, UT Southwestern Medical Center, 2Department of Microbiology, UT Southwestern Medical Center
We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (γHV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for γHV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.
JoVE General
Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry
1Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 2Deparment of Biochemistry, Research and Innovation Centre, University of Regina, 3Department of Medical Genetics and Microbiology, University of Toronto
Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.
JoVE Neuroscience
Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe
Department of Biology, University of Washington
Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.
JoVE General
Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching
1Department of Physics and Astronomy, University of Rochester, 2Department of Biomedical Engineering, University of Rochester
In this article we will describe the procedure for measuring diffusion coefficients using multi-photon fluorescence recovery after photobleaching. We will begin by aligning the laser along the optical path to the sample and determining the proper experimental parameters, then continue generating and finally fitting fluorescence recovery curves.
JoVE General
A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform
1Department of Electrical and Computer Engineering, Texas A&M University (TAMU), 2Department of Veterinary Integrative Biosciences, Texas A&M University (TAMU)
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.
JoVE General
High-throughput Screening and Biosensing with Fluorescent C. elegans Strains
1Department of Biology, University of Florida, 2Mount Desert Island Biological Laboratory
A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.
JoVE General
Procedures for Rat in situ Skeletal Muscle Contractile Properties
Faculty of Kinesiology, University of Calgary
This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle’s contractile response at 37 degrees C with an intact circulation.
JoVE General
Analysis of Cell Cycle Position in Mammalian Cells
1Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, 2London Regional Cancer Program, Children's Health Research Institute, and Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario
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.
JoVE Bioengineering
Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries
Department of Chemical and Biological Engineering, Iowa State University
This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.
JoVE General
Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging
Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.
JoVE Neuroscience
Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis
1Research Service, Veterans Administration Medical Center, Memphis, TN, 2Department of Neurology, University of Tennessee Health Science Center, Memphis, TN, 3Department of Anatomy/Neurobiology, University of Tennessee Health Science Center, Memphis, TN
A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells.
JoVE Neuroscience
In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons
1Unit on Neural Circuits and Adaptive Behaviors, Genes Cognition and Psychosis Program, National Institute of Mental Health, 2Department of Neuroscience, Brown University - National Institutes of Health Graduate Partnership Program, 3Section on Synaptic Pharmacology, Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, 4Champalimaud Neuroscience Programme, Champalimaud Center for the Unknown
Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.
JoVE General
Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction
1San Diego Regenerative Medicine Institute, 2Xcelthera, 3Department of Neurosurgery, Harvard Medical School, 4Division of SCI Research, VA Boston Healthcare System, 5Program in Stem Cell & Regenerative Biology, Sanford-Burnham Medical Research Institute, 6La Jolla IVF
We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.
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