JoVE Bioengineering
Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers
Department of Chemical Engineering, Stanford University
In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.
JoVE Bioengineering
A Microfluidic-based Hydrodynamic Trap for Single Particles
1Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 2Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign
In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.
JoVE General
Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation
1Brigham and Women's Hospital / Harvard Medical School, Department of Medicine, Cardiovascular Division, 2Weill Institute for Cell and Molecular Biology & Department of Biomedical Engineering, Cornell University
We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.
JoVE Bioengineering
Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma
Department of Mechanical and Aerospace Engineering, The George Washington University
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.
JoVE Bioengineering
Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness
1Section of Respiratory, Critical Care and Sleep Medicine, Department of Medicine, University of Illinois, 2Institute for Medicine and Engineering, University of Pennsylvania
Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.
JoVE General
Isolation and Biophysical Study of Fruit Cuticles
1Department of Chemistry, City College of New York, City University of New York Graduate Center and Institute for Macromolecular Assemblies, 2Department of Chemical Engineering, City College of New York
Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.
JoVE General
Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography
Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.
JoVE General
Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies
Department of Biology, University of Kentucky, Lexington
We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.
JoVE General
A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA
1Department of Molecular and Cellular Biology, Brown University, 2Center for Computational Molecular Biology, Brown University
Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.
JoVE General
Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping
Schulich Faculty of Chemistry, The Technion, Israel Institute of Technology
This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.
JoVE Neuroscience
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
Department of Molecular and Cellular Biology, University of Guelph
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.
JoVE General
Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs
Department of Chemical Engineering and Materials Science, City of Hope Cancer Center
Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.
JoVE Bioengineering
Monitoring Protein Adsorption with Solid-state Nanopores
Department of Physics, Syracuse University
A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.
JoVE Neuroscience
Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe
1Center for Integrative Genomics, University of Lausanne, 2Department of Biology, University of Konstanz
We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.
JoVE Neuroscience
Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation
1Department of Biochemistry and Molecular Biology, Hotchkiss Brain Institute, Alberta Children’s Hospital Research Institute, University of Calgary, 2Department of Medical Genetics, Alberta Children’s Hospital Research Institute, Hotchkiss Brain Institute, University of Calgary
In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.
JoVE Immunology and Infection
Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens
1Department of Molecular Cell Biology, Institute of Biology, Leiden University, 2Department of Medical Microbiology and Infection Control, VU University Medical Center, 3Australian Regenerative Medicine Institute, Monash University
Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.
JoVE Immunology and Infection
Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae
Department of Molecular and Biomedical Sciences, University of Maine
The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.
JoVE Immunology and Infection
IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation
1Center for Biological Security, Robert Koch-Institute, 2CICVyA - INTA Castelar, Instituto de Virología, 3Center of Molecular Immunology, Ciudad de la Habana, Cuba, 4Department of Biology, Chemistry, Pharmacy, Institute of Biology-Neurobiology, Free University of Berlin, 5Institut of Pharmacology, Charité-University Medicine of Berlin
This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.
JoVE General
Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates
1Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, 2Centre for Biological Signalling Studies (bioss) and Biology III, Faculty of Biology, University of Freiburg, 3Department of Molecular Immunology, Max-Planck-Institute of Immunology and Epigenetics
In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.
JoVE General
Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber
1Department of Physiology and Biophysics, Confocal Microscopy and Cell Imaging Core, Robert Wood Johnson Medical School, 2Department of Physiology and Biophysics, Robert Wood Johnson Medical School, 3Muscle Biology Research Group-MUBIG Schools of Nursing & Medicine, University of Missouri-Kansas City
The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.
JoVE Bioengineering
Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers
1LSA Biophysics, University of Michigan, 2LSA Biophysics, Department of Physics, University of Michigan
We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules. By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm.
JoVE Bioengineering
Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation
1Electrical Engineering Department, University of Washington, 2Division of Human Biology, Fred Hutchinson Cancer Research Center, 3Molecular and Cellular Biology Program, University of Washington, 4Clinical Research, Fred Hutchinson Cancer Research Center, 5Public Health Sciences, Fred Hutchinson Cancer Research Center
Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.
JoVE General
Using Laser Tweezers For Manipulating Isolated Neurons In Vitro
This video describes the manipulation of cultured neurons using laser tweezers in vitro.
JoVE General
Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy
1Biochemistry, University of Oxford, 2Physics, University of Oxford
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.
JoVE Immunology and Infection
Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means
1Centro de Biología Celular y Molecular de Enfermedades, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP), 2Acharya Nagarjuna University, 3Departamento de Medios y Creativo, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP)
The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.
JoVE General
Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation
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.
JoVE Bioengineering
Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow
1Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, 2Micro and Nanotechnology Lab, University of Illinois at Urbana-Champaign, 3Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, 4Bioengineering, University of Illinois at Urbana-Champaign
Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.
JoVE Applied Physics
Construction of a High Resolution Microscope with Conventional and Holographic Optical Trapping Capabilities
Department of Physics and Astronomy, University of Utah
The system described herein employs a traditional optical trap as well as an independent holographic optical trapping line, capable of creating and manipulating multiple traps. This allows for the creation of complex geometric arrangements of refractive particles while also permitting simultaneous high-speed, high-resolution measurements of the activity of biological enzymes.
JoVE General
ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection
Department of Sciences and Chemical Technologies, University of Rome, Tor Vergata
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.
JoVE General
Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2
Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology
Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.
JoVE Clinical and Translational Medicine
Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures
1Cardiology, Robotic Cardiac Electrophysiology and Arrhythmia Unit, La Paz University Hospital, 2Magnetecs Corp., 3Cardiology, Geffen School of Medicine at UCLA Los Angeles
This report provides a detailed description of a new remote navigation system based on magnetic driven forces, which has been recently introduced as a new robotic tool for human cardiac electrophysiology procedures.
JoVE General
Dissection of Drosophila Ovaries
JoVE General
Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 3
Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology
Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.
JoVE Clinical and Translational Medicine
The Polyvinyl Alcohol Sponge Model Implantation
1Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, 2The Department of Veterans Affairs Medical Center, 3Internal Medicine, Vanderbilt University School of Medicine
A useful tool to analyze the effects of drugs, growth factors, and/or manipulated cells in an animal model of wound repair is described. This technique utilizes the properties of a polyvinyl alcohol (PVA) sponge to deliver and contain the desired treatment and also provide a platform to be excised and analyzed.
JoVE Immunology and Infection
Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells
Department of Microbiology and Immunology, University of Maryland
Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.
JoVE General
Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)
1Genome Technology Branch, National Human Genome Research Institute, 2Neuroscience and Cognitive Science Program, University of Maryland
The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.
JoVE General
Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies
Groupe PERSEUS, Faculté de Génie Département de génie mécanique, Université de Sherbrooke
This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
JoVE Applied Physics
Optical Trapping of Nanoparticles
Electrical and Computer Engineering, University of Victoria
The following setup approach details low power optical trapping of dielectric nanoparticles using a double-nanohole in metal film.
JoVE General
Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting
1Department of Molecular and Microbiology, George Mason University, 2Krasnow Institute for Advanced Study, George Mason University
In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.
JoVE General
Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography
1Department of Cell Biology, Emory University, 2Department of Medicine, Division of Cardiology, Emory University
The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.
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 Neuroscience
A Novel Approach for Documenting Phosphenes Induced by Transcranial Magnetic Stimulation
1Department of Anatomy and Neurobiology, Boston University School of Medicine, 2Department of Neurology, Beth Israel Deaconess Med Center, 3Centre de Recherche de l'institut du Cerveau et la Moelle Epinière (CRICM), Centre National de la Recherche Scientifique (CNRS)
Phosphenes are transient percepts of light that can be induced by applying Transcranial Magnetic Stimulation (TMS) to visually sensitive regions of cortex. We demonstrate a standard protocol for determining the phosphene threshold value and introduce a novel method for quantifying and analyzing perceived phosphenes.
JoVE General
A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example
In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.
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 Immunology and Infection
Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells
This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.
JoVE General
PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins
1Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, Rockefeller University, 2Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, 3Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 4Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 5Genomics Resource Center, Rockefeller University
RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.
JoVE Clinical and Translational Medicine
The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice
1The Lundbeck Foundation Research Center MIND, Department of Biomedicine, Aarhus University, 2Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen
The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.
JoVE General
Isolation and Culture of Avian Embryonic Valvular Progenitor Cells
Department of Biomedical Engineering, Cornell University
This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.
JoVE General
Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy
1Biosphere Oriented Biology Research Unit, RIKEN Advanced Science Institute, 2Graduate School of Nanobioscience, Yokohama City University, 3Advanced NMR Metabomics Research Team, RIKEN Plant Science Center, 4Graduate School of Bioagricultural Science, Nagoya University
A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.
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).
More Results...
