JoVE General
Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues
1Department of Horticulture and Crop Science, The Ohio State University, 2Department of Plant Pathology, North Carolina State University
We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.
JoVE General
Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens
Department of Biology and Biotechnology, Worcester Polytechnic Institute- WPI
A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1.
JoVE General
A β-glucuronidase (GUS) Based Cell Death Assay
Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.
JoVE General
Generation of Transgenic C. elegans by Biolistic Transformation
Department of Medicine, University of Pittsburgh
Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.
JoVE General
Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun
Dept. Of Cell Biology and Molecular Genetics, University of Maryland
This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)
JoVE Applied Physics
Characterization of Surface Modifications by White Light Interferometry: Applications in Ion Sputtering, Laser Ablation, and Tribology Experiments
1Materials Science Division, Argonne National Laboratory, 2Energy Systems Division, Argonne National Laboratory, 3MassThink LLC
White light microscope interferometry is an optical, noncontact and quick method for measuring the topography of surfaces. It is shown how the method can be applied toward mechanical wear analysis, where wear scars on tribological test samples are analyzed; and in materials science to determine ion beam sputtering or laser ablation volumes and depths.
JoVE General
A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment
1Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, 2Bio-Medical Engineering Department, NED University of Engineering and Technology
Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.
JoVE General
Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture
Center for Neuroscience, University of California, Davis
We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.
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
Whole-Body Nanoparticle Aerosol Inhalation Exposures
1Department of Physiology and Pharmacology, School of Medicine, West Virginia University, 2Center for Cardiovascular and Respiratory Sciences, West Virginia University, 3National Institute for Occupational Safety and Health
A whole-body nanoparticle aerosol inhalation exposure facility was constructed for nano-sized titanium dioxide (TiO2) inhalation toxicology studies. This system provides nano-TiO2 aerosol test atmospheres that have: 1) a steady mass concentration; 2) a homogenous composition free of contaminants; and 3) a stable particle size distribution during aerosol generation.
JoVE Bioengineering
Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry
1Biomedical Engineering Department, Johns Hopkins University School of Medicine, 2Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, 3Wilmer Eye Institute, Johns Hopkins University School of Medicine, 4Institute for Nanobiotechnology, Johns Hopkins University School of Medicine
A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.
JoVE Immunology and Infection
Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging
1Experimental and Clinical Research Center, A joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine, 2Berlin Ultrahigh Field Facility (B.U.F.F.), Max Delbrück Center for Molecular Medicine
Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.
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 Bioengineering
Viral Nanoparticles for In vivo Tumor Imaging
1Department of Biomedical Engineering, Case Western Reserve University, 2Department of Biomedical Engineering, Radiology, and Materials Science and Engineering, Case Western Reserve University
Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.
JoVE General
The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker
1Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Department of Medicine, Division of Geriatric Medicine and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh
The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.
JoVE Applied Physics
Compact Quantum Dots for Single-molecule Imaging
1Department of Biomedical Engineering, Emory University, 2Department of Chemistry, Georgia Institute of Technology
We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.
JoVE General
Localized RNAi and Ectopic Gene Expression in the Medicinal Leech
1Division of Biological Sciences, University of California San Diego - UCSD, 2Department of Physics, University of California San Diego - UCSD
In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.
JoVE Immunology and Infection
Particle Agglutination Method for Poliovirus Identification
1Department of Virology II, National Institute of Infectious Diseases, 2New Product Design Department, Fujirebio Inc.
A recently developed novel particle agglutination (PA) assay utilizing virus receptor molecule allowed a rapid and easy identification of poliovirus (PV). In this article, we will show the procedure for the PA assay for PV identification.
JoVE Neuroscience
Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures
1Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 2Department of Microbiology and Molecular Genetics, Medical College of Wisconsin
The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.
JoVE General
Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources
Max-Planck Institute for Evolutionary Anthropology, Leipzig
We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.
JoVE General
Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)
1Department of Biochemistry / Analytics, caprotec bioanalytics GmbH, 2Institute of Organic Chemistry, RWTH Aachen University
Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.
JoVE Immunology and Infection
Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles
Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.
JoVE General
Annotation of Plant Gene Function via Combined Genomics, Metabolomics and Informatics
Molekulare Pflanzenphysiologie, Max-Planck-Institut
Combination of genomics, co-expression gene analysis and the identification of target compounds via metabolism give gene functional annotation.
JoVE Applied Physics
Encapsulation and Permeability Characteristics of Plasma Polymerized Hollow Particles
Department of Chemical Engineering, The Pennsylvania State University
We have used plasma enhanced chemical vapor deposition to deposit thin films ranging from a few nm to several 100 nm on nano-sized particles of various materials. We subsequently etch the core material to produce hollow nanoshells whose permeability is controlled by the thickness of the shell. We characterize the permeability of these coatings to small solutes and demonstrate that these barriers can provide sustained release of the core material over several days.
JoVE Applied Physics
Echo Particle Image Velocimetry
Mechanical Engineering Department, University of New Hampshire
An echo particle image velocimetry (EPIV) system capable of acquiring two-dimensional fields of velocity in optically opaque fluids or through optically opaque geometries is described, and validation measurements in pipe flow are reported.
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 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 Bioengineering
Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus (SCUVA)
1Applied Ocean Physics and Engineering, Woods Hole Oceanographic Institution, 2Environmental Science and Marine Biology, Roger Williams University, 3Marine Biology Laboratory, Whitman Center, 4Department of Biology, Providence College, 5Departments of Aeronautics and Bioengineering, California Institute of Technology
This protocol provides instructions on how to use a self-contained underwater velocimetry apparatus (SCUVA), which is designed for quantification of in situ animal-generated flows. In addition, this protocol addresses challenges posed by field conditions, and includes operator motion, predicting position of animals, and orientation of SCUVA.
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
RNAi Screening to Identify Postembryonic Phenotypes in C. elegans
Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center
We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.
JoVE Bioengineering
High Throughput Single-cell and Multiple-cell Micro-encapsulation
Department of Mechanical Engineering, Vanderbilt University
Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.
JoVE General
Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method
Molecular Biophysics and Biochemistry, Yale University
This article describes a standard method to get a three-dimensional (3D) reconstruction of biological macromolecules using negative staining electron microscopy (EM). In this protocol, we explain how to get the 3D structure of the Saccharomyces cerevisiae exosome complex at medium resolution using the random conical tilt reconstruction method (RCT).
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 Bioengineering
Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows
1Department of Chemical and Biological Engineering, University of Ottawa, 2Department of Mechanical Engineering, University of Ottawa
Micro-particle image velocimetry (μPIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.
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 General
Generation of Mice Derived from Induced Pluripotent Stem Cells
1Dorris Neuroscience Center & Department of Cell Biology, The Scripps Research Institute, 2Mouse Genetics Core Facility, The Scripps Research Institute
Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.
JoVE Chemistry
Preparation and Use of Samarium Diiodide (SmI2) in Organic Synthesis: The Mechanistic Role of HMPA and Ni(II) Salts in the Samarium Barbier Reaction
Department of Chemistry, Lehigh University
A straightforward procedure for the preparation of samarium diiodide (SmI2) in THF is described. The role of two main additives namely hexamethylphosphoramide (HMPA) and Ni(acac)2 in Sm mediated reactions is demonstrated in the Sm-Barbier reaction.
JoVE General
Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter
1Orflo Technologies, 2University of Utah
The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.
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 General
AC Electrokinetic Phenomena Generated by Microelectrode Structures
1Biomedical Engineering, Science and Health Systems, Drexel University, 2Mechanical Engineering and Mechanics, Drexel University
Manipulating fluids and suspended particles in the micro- and nano-scale is becoming more of a reality as enabling technologies, like AC electrokinetics, continue to develop. Here, we discuss the physics behind AC electrokinetics, how to fabricate these devices and how to interpret the experimental observations.
JoVE Bioengineering
Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution
Department of Mechanical, Aerospace, and Nuclear Engineering, Rensselaer Polytechnic Institute
The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.
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 General
IP-FCM: Immunoprecipitation Detected by Flow Cytometry
Department of Immunology, College of Medicine, Mayo Clinic
The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.
JoVE General
Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice
Blood Research Institute, BloodCenter of Wisconsin
Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.
JoVE General
High-throughput Purification of Affinity-tagged Recombinant Proteins
Department of Life Sciences, Imperial College London
We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.
JoVE Immunology and Infection
Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction
Department of Structural Biology, University of Pittsburgh School of Medicine
This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.
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 Bioengineering
Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy
1College of Nursing, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 2College of Science and Engineering, Interdisciplinary Life Sciences Research Laboratory, Seattle University
A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.
JoVE Immunology and Infection
Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging
1Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, 2Department of Mechanical and Aerospace Engineering, The Ohio State University, 3Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, 4Dept. of Chemical and Biomolecular Engineering, Vanderbilt University
A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.
JoVE Neuroscience
Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice
1Laboratory of Experimental Hematology, University of Antwerp, 2Bio Imaging Lab, University of Antwerp
This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.
More Results...
