JoVE Bioengineering
Cell Co-culture Patterning Using Aqueous Two-phase Systems
1Department of Biomedical Engineering, University of Michigan, 2Department of Macromolecular Science and Engineering, University of Michigan
Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.
JoVE Bioengineering
Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique
1Laboratory of Nano- and Translational Medicine, Department of Radiation Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, 2Carolina Center for Nanotechnology Excellence, University of North Carolina
This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.
JoVE Bioengineering
Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography
Biomedical Engineering, Tulane University
Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.
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 General
Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes
Department of Biomedical Engineering, University of California, Davis
Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.
JoVE Bioengineering
Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles
Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University
Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.
JoVE Bioengineering
Generation and Recovery of β-cell Spheroids From Step-growth PEG-peptide Hydrogels
The following protocol provides techniques for encapsulating pancreatic β-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.
JoVE Bioengineering
Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy
1Centre for Vascular Research and Australian Centre for Nanomedicine, The University of New South Wales, 2School of Chemistry and Australian Centre for Nanomedicine, The University of New South Wales
A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.
JoVE Bioengineering
Tracking Hypoxic Signaling within Encapsulated Cell Aggregates
1Biomedical Engineering Program, University of South Carolina, 2Chemical Engineering Department, University of South Carolina
A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.
JoVE Bioengineering
Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering
1Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 2Biomedical Sciences Program, University of California, San Diego, 3Department of Bioengineering, University of California, San Diego
Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues
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 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 Immunology and Infection
Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors
1Division of Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research, 2Aaron Diamond AIDS Research Center, Laboratory of Retrovirology, The Rockefeller University, NY
We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.
JoVE Clinical and Translational Medicine
Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging
1Department of Medical Biophysics, University of Western Ontario, 2London Regional Cancer Program, London Health Science Centre, 3Department of Pathology, Vanderbilt University, 4Translational Prostate Cancer Research Group, London Health Science Centre
We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.
JoVE General
Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules
1Department of Physics, Texas A&M University (TAMU), 2Department of Biomedical Engineering, Texas A&M University (TAMU)
We present a procedure for forming a poly(ethylene glycol) self-assembled monolayer (PEG-SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single step and prevents biofouling on silicon and gold surfaces. Electrophoresis is then used for patterning biomolecules down to the nanoscale.
JoVE General
Visualizing Single-molecule DNA Replication with Fluorescence Microscopy
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School
This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.
JoVE General
Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay
We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.
JoVE General
Heterokaryon Technique for Analysis of Cell Type-specific Localization
Department of Chemistry and Biochemistry, Worcester Polytechnic Institute- WPI
A flexible and efficient method for the characterization of cell type-specific protein localization and nucleocytoplasmic shuttling is described. This heterokaryon approach uses fluorescently-labeled fusion proteins to image protein localizations after cell fusion. The protocol is amenable to steady-state localizations or more dynamic determinations based on live cell imaging.
JoVE General
High-throughput Yeast Plasmid Overexpression Screen
1Neuroscience Graduate Group, University of Pennsylvania School of Medicine, 2Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine
Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.
JoVE Immunology and Infection
Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols
1Centre for Blood Research, University of British Columbia, 2Department of Pathology and Laboratory Medicine, University of British Columbia, 3Canadian Blood Services, University of British Columbia, 4Department of Chemistry, Life Sciences Centre, University of British Columbia
The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.
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 Bioengineering
Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns
1Department of Materials Science and Engineering, MIT - Massachusetts Institute of Technology, 2Department of Mechanical Engineering, MIT - Massachusetts Institute of Technology, 3HST Center for Biomedical Engineering and Harvard Stem Cell Institute, Brigham and Women's Hospital and Harvard Medical School
We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.
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 Applied Physics
Micro 3D Printing Using a Digital Projector and its Application in the Study of Soft Materials Mechanics
Department of Mechanical Engineering, Massachusetts Institute of Technology
We demonstrate controlled pattern transformation of swelling gel tubes by elastic instability. A simple projection micro stereo-lithography setup is built using an off-the-shelf digital data projector to fabricate three-dimensional polymeric structures in a layer-by-layer fashion. Swelling hydrogel tubes under mechanical constraint display various circumferential buckling modes depending on dimension.
JoVE Editorial
December 2012: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here are some highlights from the December 2012 Issue of Journal of Visualized Experiments (JoVE).
JoVE Neuroscience
Electrophysiology of Scorpion Peg Sensilla
Department of Zoology, University of Oklahoma
This article describes an electrophysiological method for isolating chemical stimulation to individual sensilla via extracellular, tip-recordings under mineral oil.
JoVE General
Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems
Department of Biochemistry and Biophysics, University of Pennsylvania
We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.
JoVE Neuroscience
Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae
Laboratory of Neurogenetics and Behavior, Rockefeller University
Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).
JoVE Immunology and Infection
Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs
1Plant Sciences Institute, Agricultural Research Service, United States Department of Agriculture, 2Molecular Plant Pathology Laboratory, Agricultural Research Service, United States Department of Agriculture
A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.
JoVE General
Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions
1Department of Biochemistry, University of Toronto, 2Department of Molecular Genetics, University of Toronto, 3Terrence Donnelly Centre for Cellular and Biomolecular Research (CCBR), University of Toronto
MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.
JoVE General
Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin
1Department of Orthopaedic Surgery, NYU Hospital for Joint Diseases, 2Department of Cell Biology, New York University School of Medicine
We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.
JoVE General
Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein
1Molecular and Cell Biology, Institute for Biomedical Aging Research, 2Department of Gerontology and Geriatrics, Netherlands Consortium for Healthy Aging, Leiden University Medical Center
A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.
JoVE General
Protein Crystallization for X-ray Crystallography
Molecular Biochemistry and Biophysics, Yale University
The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.
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 Editorial
April 2011: This Month in JoVE
Here are some highlights from the April 2011 Issue of Journal of Visualized Experiments (JoVE).
JoVE Bioengineering
Microfabricated Platforms for Mechanically Dynamic Cell Culture
1Department of Mechanical and Industrial Engineering, University of Toronto, 2Institute of Biomaterials and Biomedical Engineering, University of Toronto, 3Faculty of Dentistry, University of Toronto
In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.
JoVE General
Extraction of High Molecular Weight DNA from Microbial Mats
We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.
JoVE Bioengineering
Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells
A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.
JoVE General
DNA Stable-Isotope Probing (DNA-SIP)
Department of Biology, University of Waterloo
DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.
JoVE Applied Physics
Synthesis of Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation
Department of Mechanical Engineering, Boston University
Phase-shift nanoemulsions (PSNE) can be vaporized using high intensity focused ultrasound to enhance localized heating and improve thermal ablation in tumors. In this report, the preparation of stable PSNE with a narrow size distribution is described. Furthermore, the impact of vaporized PSNE on ultrasound-mediated ablation is demonstrated in tissue-mimicking phantoms.
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
Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System
Laboratory of Molecular Gerontology, National Institute on Aging, NIH
Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.
JoVE General
Cellular Encapsulation in 3D Hydrogels for Tissue Engineering
1Department of Bioengineering, University of Pennsylvania, 2Department of Bioengineering, University of Pennsylvania-School of Medicine
We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.
JoVE Bioengineering
Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems
1Bioengineering Department, Northeastern University, 2Department of Pharmaceutical Sciences, Northeastern University
Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.
JoVE General
Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects
1Orthopaedics Research, University of Virginia, 2Biological Sciences, University of Delaware, 3Orthopaedic Surgery, University of Virginia
A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.
JoVE General
DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning
Department of Pharmacology, University of California, Davis
We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.
JoVE General
Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast
Department of Plant Sciences, Tel Aviv University
In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.
JoVE Bioengineering
In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage
1Neurosurgery, Baylor College of Medicine, 2Michael E. DeBakey Veterans Affairs Medical Center, 3Molecular & Cellular Biology, Baylor College of Medicine
We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.
JoVE General
Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules
1Physics Department, Boston University, 2Department of Biomedical Engineering, Boston University
In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.
JoVE General
Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time
1Mechanical Engineering, Johns Hopkins University, 2Biomedical Engineering, Duke University, 3Biomedical Engineering, Johns Hopkins University
We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.
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