JoVE General
Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse
Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.
JoVE Immunology and Infection
Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers
1Department of Pathology, New York University Langone School of Medicine, 2Program in Molecular Pathogenesis, Marty and Helen Kimmel Center for Biology and Medicine and Skirball Institute for Biomolecular Medicine, 3Laboratory of Molecular Immunogenetics, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 4Veteran Affairs New York Harbor Healthcare System
This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.
JoVE General
Method for Measurement of Viral Fusion Kinetics at the Single Particle Level
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.
JoVE General
Preparation of Artificial Bilayers for Electrophysiology Experiments
Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University
Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.
JoVE Immunology and Infection
A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins
The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.
JoVE Chemistry
Origami Inspired Self-assembly of Patterned and Reconfigurable Particles
1Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 2Department of Chemistry, The Johns Hopkins University
We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.
JoVE Bioengineering
Graphene Coatings for Biomedical Implants
1Department of Physics, Clemson University, 2Department of Pharmacology and Toxicology, East Carolina University, 3Department of Bioengineering, Clemson University, 4Center for Optical Materials Science and Engineering Technologies, Clemson University
Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.
JoVE General
Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties
Department of Physiology and Biophysics, Weill Cornell Medical College
We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.
JoVE General
Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures
Bioengineering, University of Illinois
Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.
JoVE Applied Physics
Polycrystalline Silicon Thin-film Solar cells with Plasmonic-enhanced Light-trapping
School of Photovoltaics, University of New South Wales
Polycrystalline silicon thin-film solar cells on glass are fabricated by deposition of boron and phosphorous doped silicon layers followed by crystallisation, defect passivation and metallisation. Plasmonic light-trapping is introduced by forming Ag nanoparticles on the silicon cell surface capped with a diffused reflector resulting in ~45% photocurrent enhancement.
JoVE General
Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties
1Department of Physiology and Biophysics, Weill Cornell Medical College, 2Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University
Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.
JoVE Immunology and Infection
Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs
1Department of Biomedical Engineering, Vanderbilt University, 2Vanderbilt Institute for Nanoscale Science & Engineering, Vanderbilt University, 3Interdisciplinary Materials Science Program, Vanderbilt University, 4Monroe Carell Jr. Children's Hospital, Vanderbilt University Medical Center, 5Department of Chemical & Biomolecular Engineering, Vanderbilt University, 6Department of Cancer Biology, Vanderbilt University
A hemolysis assay can be used as a rapid, high-throughput screen of drug delivery systems' cytocompatibility and endosomolytic activity for intracellular cargo delivery. The assay measures the disruption of erythrocyte membranes as a function of environmental pH.
JoVE Bioengineering
Planar and Three-Dimensional Printing of Conductive Inks
1Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, 2Center for Micro- and Nanotechnology, Lawrence Livermore National Laboratory, 3Presently at the Interdisciplinary Center for Wide Band-gap Semiconductors, University Of California Santa Barbara
Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.
JoVE Bioengineering
Postproduction Processing of Electrospun Fibres for Tissue Engineering
1Materials Science and Engineering, University of Sheffield, 2Department of Biomedical Science, University of Sheffield, 3Department of Chemistry, University of Sheffield
Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.
JoVE General
High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method
1UCLA-DOE Institute for Genomics and Proteomics, University of California Los Angeles, 2Department of Physiology, David Geffen School of Medicine, UCLA
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.
JoVE General
Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine
A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.
JoVE General
A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles
Department of Biochemistry and Molecular Biology, University of British Columbia
Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.
JoVE General
Microcontact Printing of Proteins for Cell Biology
Department of Biomedical Engineering, Columbia University
Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.
JoVE Neuroscience
Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures
Section on Critical Brain Dynamics, National Institute of Mental Health
A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.
JoVE Bioengineering
Engineering a Bilayered Hydrogel to Control ASC Differentiation
1Department of Extremity Trauma Research and Regenerative Medicine, United States Army Institute of Surgical Research, 2Department of Biomedical Engineering, The University of Texas at Austin
This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.
JoVE Neuroscience
Neural Tube Closure in Mouse Whole Embryo Culture
Department of Neurology/Neuroscience, Weill Cornell Medical College
A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.
JoVE General
Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila
1Center for Systems Biology, Massachusetts General Hospital, 2Institute for Biological and Medical Imaging (IBMI), Technical University of Munich and Helmholtz Center Munich, 3Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute
Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.
JoVE General
Measuring Peptide Translocation into Large Unilamellar Vesicles
1Department of Chemistry, Wellesley College,
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.
JoVE General
Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation
1Institute for Biological Interfaces, Karlsruhe Research Centre, 2Institute for BioMedical Technology, University of Twente, 3Department of Materials Research, Institute for Heavy Ion Research, 4Institute of Microstructure Technology, Karlsruhe Research Centre, 5Institute for Micro Process Engineering, Karlsruhe Research Centre
We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.
JoVE Editorial
December 2011: This Month in JoVE
Here are some highlights from the December 2011 Issue of Journal of Visualized Experiments (JoVE).
JoVE Bioengineering
Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy
Chemistry and Biochemistry, University of California San Diego - UCSD
This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.
JoVE Neuroscience
The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow
1Department of Neurosurgery, The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco - UCSF, 2Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, 3Department of Neuroscience and Neurology, College of Physicians and Surgeons, Columbia University, 4Department of Developmental and Regenerative Biology, Nagoya City University Graduate School of Medical Sciences, 5Center for Motor Neuron Biology and Disease, College of Physicians and Surgeons, Columbia University
The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.
JoVE Neuroscience
Functional Mapping with Simultaneous MEG and EEG
Athinoula A. Martinos Center for Biomedical Imaging, MGH - Massachusetts General Hospital
We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli.
JoVE General
Microfluidic Chips Controlled with Elastomeric Microvalve Arrays
Dept. of Bioengineering, University of Washington
We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.
JoVE General
Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer
Department of Chemistry and Biochemistry, Southern Illinois University
We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.
JoVE Bioengineering
Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone
1Electrical Engineering Department, University of California, Los Angeles, 2Bioengineering Department, University of California, Los Angeles, 3California NanoSystems Institute (CNSI), University of California, Los Angeles
We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.
JoVE Neuroscience
Multielectrode Array Recordings of the Vomeronasal Epithelium
Anatomy and Neurobiology, Washington University School of Medicine
Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.
JoVE General
Procedure for Fabricating Biofunctional Nanofibers
1Department of Chemistry, Clark Atlanta University, 2Department of Physics, Clark Atlanta University, 3Department of Chemistry and Chemical Biology, Cornell University
An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.
JoVE Immunology and Infection
Generation of Recombinant Influenza Virus from Plasmid DNA
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Departments of Microbiology and Medicine, and Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine
Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.
JoVE Neuroscience
Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals
1Biomedical Engineering, University of Michigan, 2Biomedical Engineering, University of Wisconsin-Madison, 3NeuroNexus Technologies
We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.
JoVE Bioengineering
Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells
1CYTOO Cell Architects, Grenoble, France, 2Centre Commun de Quantimétrie, Faculté de Médecine Rockefeller, Lyon, France
Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.
JoVE Bioengineering
Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth
1Department of Biological Systems Engineering, University of Nebraska-Lincoln, 2Department of Engineering Mechanics, University of Nebraska-Lincoln
The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (μMRE).
JoVE Editorial
August 2011: This Month in JoVE
Here are some highlights from the August 2011 Issue of Journal of Visualized Experiments (JoVE).
JoVE Bioengineering
Magnetically-Assisted Remote Controlled Microcatheter Tip Deflection under Magnetic Resonance Imaging
1Department of Radiology and Biomedical Imaging, University of California, San Francisco, 2School of Medicine, University of California, San Francisco, 3Department of Radiology and Biomedical Imaging, UCSF Medical Center, 4University of California, San Francisco, 5Hansen Medical, Mountain View, CA
Current applied to an endovascular microcatheter with microcoil tip made by laser lathe lithography can achieve controllable deflections under magnetic resonance (MR) guidance, which may improve speed and efficacy of navigation of vasculature during various endovascular procedures.
JoVE General
Imaging Cell Shape Change in Living Drosophila Embryos
1Program in Cell & Molecular Biology, Baylor College of Medicine (BCM), 2Verna & Marrs McLean Department of Biochemistry & Molecular Biology, Baylor College of Medicine (BCM)
Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.
JoVE Neuroscience
Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging
1Center for the Neural Basis of Cognition, 2Department of Psychology, University of Pittsburgh, 3Department of Psychology, Carnegie Mellon University, 4Department of Bioengineering, University of Pittsburgh
We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.
JoVE Chemistry
Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies
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.
JoVE General
Measuring the Induced Membrane Voltage with Di-8-ANEPPS
Department of Biomedical Engineering, Faculty of Electrical Engineering, University of Ljubljana
External electric field induces a voltage on the membrane of a cell, termed the induced membrane voltage (ΔΦ). By using the potentiometric dye di-8-ANEPPS, it is possible to measure the ΔΦ noninvasively. This video shows the protocol for measuring ΔΦ using di-8-ANEPPS.
JoVE General
Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging
This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.
JoVE Clinical and Translational Medicine
Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture
Life Science Division, Lawrence Berkeley National Laboratory
A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.
JoVE Clinical and Translational Medicine
Probing the Brain in Autism Using fMRI and Diffusion Tensor Imaging
Department of Psychology, University of Alabama at Birmingham
Neuroimaging techniques, such as functional MRI and Diffusion Tensor Imaging have become increasingly useful in characterizing the cognitive and neural deficits in autism. An examination of brain connectivity in autism at a network level along with adaptations for scanning children with developmental disabilities is presented.
JoVE Clinical and Translational Medicine
An Investigation of the Effects of Sports-related Concussion in Youth Using Functional Magnetic Resonance Imaging and the Head Impact Telemetry System
1Graduate Department of Rehabilitation Science, University of Toronto, 2Occupational Science and Occupational Therapy, University of Toronto, 3Department of Psychology, University of Toronto, 4Bloorview Kids Rehab, 5Toronto Rehab, 6Cognitive Neurology, Sunnybrook Health Sciences Centre, 7Faculty of Medicine, University of Toronto
This article provides an overview of a multi-modal approach to mild traumatic brain injury diagnosis and recovery in youth. This approach combines neuropsychological testing with functional magnetic resonance imaging and the Head Impact Telemetry System to monitor the relationship between head impacts and brain activity during cognitive testing.
JoVE Immunology and Infection
Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins
1Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, 2Research service, 151, Veterans Affairs Greater Los Angeles Healthcare System, 3Departments of Medicine, Urology at David Geffen School of Medicine and Department of Microbiology, Immunology and Molecular Gentics, University of California Los Angeles (UCLA), 4Division of Infectious Diseases, 111F, Veterans Affairs Greater Los Angeles Health Care System
An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.
JoVE General
Transmembrane Domain Oligomerization Propensity determined by ToxR Assay
Department of Chemistry and Biochemistry, University of Colorado at Boulder
An efficient procedure to assess the oligomerization propensity of single-pass transmembrane domains (TMDs) is described. Chimeric proteins consisting of the TMD fused to ToxR are expressed in an E. coli reporter strain. TMD-induced oligomerization causes dimerization of ToxR, activation of transcription and production of the reporter protein, -galactosidase.
JoVE Neuroscience
Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays
Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.
More Results...
