JoVE Applied Physics
Controlling the Size, Shape and Stability of Supramolecular Polymers in Water
1Organic Chemistry Institute and CeNTech, Westfälische Wilhelms-Universität Münster, 2Laboratory of Macromolecular and Organic Chemistry, Institute for Complex Molecular Systems, Eindhoven University of Technology, 3Laboratory of Materials and Interface Chemistry and Soft Matter Research Unit, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology
The goal of this experiment is to determine and control the size, shape and stability of self-assembled discotic amphiphiles in water. For aqueous based supramolecular polymers such level of control is very difficult. We apply a strategy using both repulsive and attractive interactions. The experimental techniques applied to characterize this system are broadly applicable.
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.
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
Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering
Biomedical Engineering Department, Worcester Polytechnic Institute
This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.
JoVE Editorial
November 2011: This Month in JoVE
Here are some highlights from the November 2011 Issue of Journal of Visualized Experiments (JoVE).
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 Bioengineering
Measurement of Aggregate Cohesion by Tissue Surface Tensiometry
Department of Surgery, UMDNJ-Robert Wood Johnson Medical School
We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.
JoVE Editorial
February 2013: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here's a look at what's coming up in the February 2013 Issue of Journal of Visualized Experiments (JoVE).
JoVE Applied Physics
Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy
Materials Sciences Division, Lawrence Berkeley National Laboratory
We have developed a self-contained liquid cell, which allows imaging through liquids using a transmission electron microscope. Dynamic processes of nanoparticles in liquids can be revealed in real time with sub-nanometer resolution.
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 Applied Physics
Nanomoulding of Functional Materials, a Versatile Complementary Pattern Replication Method to Nanoimprinting
1Institute of Microengineering (IMT), Photovoltaics and Thin Film Electronics Laboratory, Ecole Polytechnique Fédérale de Lausanne (EPFL), 2Department of Electrical Engineering and Computer Sciences, University of California, Berkeley
We describe a nanomoulding technique which allows low-cost nanoscale patterning of functional materials, materials stacks and full devices. Nanomoulding can be performed on any nanoimprinting setup and can be applied to a wide range of materials and deposition processes.
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
Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets
Molecular Foundry, Lawrence Berkeley National Laboratory
A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.
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
Bridging the Bio-Electronic Interface with Biofabrication
1Fischell Department of Bioengineering, University of Maryland, 2Institute for Bioscience and Biotechnology Research, University of Maryland, 3Department of Materials Science and Engineering, University of Maryland
This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.
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
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 Bioengineering
Plasma Lithography Surface Patterning for Creation of Cell Networks
1Aerospace and Mechanical Engineering, University of Arizona, 2Biomedical Engineering IDP and BIO5 Institute, University of Arizona
A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.
JoVE General
Super-resolution Imaging of the Bacterial Division Machinery
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine
We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.
JoVE Bioengineering
Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel
Albrecht-Kossel-Institute for Neuroregeneration, University of Rostock
Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.
JoVE Bioengineering
A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids
Department of Surgery, UMDNJ-Robert Wood Johnson Medical School
We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.
JoVE Clinical and Translational Medicine
Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy
1Raymond and Beverly Sackler Foundation, 2The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, 3School of Natural Sciences, Institute for Advanced Study, Princeton, New Jersey
We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.
JoVE General
Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration
Program in Epithelial Biology, Stanford University School of Medicine
Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.
JoVE Immunology and Infection
A Visual Assay to Monitor T6SS-mediated Bacterial Competition
We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.
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 Bioengineering
High-throughput Protein Expression Generator Using a Microfluidic Platform
We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.
JoVE Neuroscience
A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats
Psychology, University of Toronto Scarborough
Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.
JoVE Neuroscience
Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography
College of Pharmacy, Division of Pharmacology and Toxicology, The University of Texas at Austin
A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.
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
Electron Cryotomography of Bacterial Cells
1Division of Biology, California Institute of Technology - Caltech, 2Howard Hughes Medical Institute, California Institute of Technology - Caltech
We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.
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
In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen
1Department of Chemistry, University of Toronto, 2Institute for Optical Sciences, University of Toronto
Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.
JoVE Bioengineering
Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method
1Department of Physics and Astronomy, The University of Texas at San Antonio, 2Centro de Investigaciones en Optica A. C., 3Department of Biology and Neurosciences Institute, The University of Texas at San Antonio
We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.
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 Bioengineering
Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry
1Department of Chemistry, Gottwald Center for the Sciences, University of Richmond, 2Department of Biochemistry and Molecular Biology, Gottwald Center for the Sciences, University of Richmond
Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).
JoVE General
Spinal Cord Electrophysiology
1The Salk Institute for Biological Studies, Howard Hughes Medical Institute and Gene Expression Laboratory, 2Biology Graduate Program, University of California San Diego - UCSD
A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.
JoVE General
Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups
Department of Chemistry and Biochemistry, University of Notre Dame
We present the high-temperature synthesis of intermetallic precursors K4Ge9, their dissolution in ethylenediamine to form Ge94- deltahedral Zintl ions, and the reaction of the clusters with alkynes to form organo-Zintl ions. The latter are characterized by electrospray mass spectrometry in solutions and by single-crystal X-ray diffraction in the solid state.
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 General
Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes
Department of Biochemistry and Biomedical Sciences, McMaster University
Crystal structure of protein–DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.
JoVE General
Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences
We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.
JoVE Neuroscience
Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration
Department of Biology, University of Victoria
We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.
JoVE General
Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species
1Department of Neuropathology, Medical School Düsseldorf, Germany, 2Center of Behavioral Neuroscience, University of Düsseldorf
The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.
JoVE Applied Physics
Harvesting Solar Energy by Means of Charge-Separating Nanocrystals and Their Solids
1Department of Physics, Bowling Green State University, 2The Center for Photochemical Sciences, Bowling Green State University, 3Department of Chemistry, Bowling Green State University
A general strategy for the development of charge-separating semiconductor nanocrystal composites deployable for solar energy production is presented. We show that assembly of donor-acceptor nanocrystal domains in a single nanoparticle geometry gives rise to a photocatalytic function, while bulk-heterojunctions of donor-acceptor nanocrystal films can be used for photovoltaic energy conversion.
JoVE General
Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations
Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg
Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.
JoVE General
RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus
Biology Department, California State University
In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.
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
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 Bioengineering
Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs
Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center
Nanoparticles of indinavir, ritonavir, efavirenz and atazanavir were manufactured using wet milling, homogenization and ultrasonication. These nanoformulations, collectively termed nanoformulated antiretroviral therapy (nanoART), assessed macrophage-based drug delivery. Monocyte-derived macrophage nanoART uptake, retention and sustained release were determined. These preliminary studies suggest the potential of nanoART for clinical use.
JoVE Bioengineering
A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments
1Department of Biotechnology, Delft University of Technology, 2Delft Center for Systems and Control, Delft University of Technology
A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via β-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.
JoVE Immunology and Infection
Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling
1Cluster of Excellence CellNetworks, Department of Infectious Diseases, Virology, Heidelberg University, 2Department of Infectious Diseases, Virology, Heidelberg University
We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.
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