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 General
Tangential Flow Ultrafiltration: A “Green” Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles
1Department of Chemistry, Wright State University, 2Department of Neuroscience, Cell Biology, and Physiology, Wright State University
Tangential flow ultrafiltration (TFU) is a recirculation method used for the weight-based separation of biosamples. TFU was adapted to size-select (1-20 nm diameter) and highly concentrate a large volume of polydisperse silver nanoparticles (4 L of 15.2 μg ml-1 down to 4 ml of 8,539.9 μg ml-1) with minimal aggregation.
JoVE General
Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions
Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute
AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.
JoVE Bioengineering
Photoacoustic Cystography
1Department of Biomedical Engineering, University at Buffalo, The State University of New York, 2Department of Creative IT Engineering, Pohang University of Science and Technology (POSTECH), 3School of Electrical Engineering and Computer Science, Kyungpook National University
Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.
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 Applied Physics
Polycrystalline Silicon Thin-film Solar cells with Plasmonic-enhanced Light-trapping
School of Photovoltaics, University of New South Wales
Polycrystalline silicon thin-film solar cells on glass are fabricated by deposition of boron and phosphorous doped silicon layers followed by crystallisation, defect passivation and metallisation. Plasmonic light-trapping is introduced by forming Ag nanoparticles on the silicon cell surface capped with a diffused reflector resulting in ~45% photocurrent enhancement.
JoVE Chemistry
Template Directed Synthesis of Plasmonic Gold Nanotubes with Tunable IR Absorbance
Department of Chemistry, University of Toronto
Solution-suspendable gold nanotubes with controlled dimensions can be synthesized by electrochemical deposition in porous anodic aluminum oxide (AAO) membranes using a hydrophobic polymer core. Gold nanotubes and nanotube arrays hold promise for applications in plasmonic biosensing, surface-enhanced Raman spectroscopy, photo-thermal heating, ionic and molecular transport, microfluidics, catalysis and electrochemical sensing.
JoVE Applied Physics
Synthesis and Functionalization of Nitrogen-doped Carbon Nanotube Cups with Gold Nanoparticles as Cork Stoppers
Department of Chemistry, University of Pittsburgh
We discussed the synthesis of individual graphitic nanocups using a series of techniques including chemical vapor deposition, acid oxidation and probe-tip sonication. By citrate reduction of HAuCl4, the graphitic nanocups were effectively corked with gold nanoparticles due to the chemically reactive edges of the cups.
JoVE General
The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis
We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.
JoVE Immunology and Infection
Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance
Department of Molecular Biology and Microbiology, University of Central Florida
In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol. This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of toxins.
JoVE Applied Physics
Synthesis and Operation of Fluorescent-core Microcavities for Refractometric Sensing
Department of Physics, University of Alberta
Fluorescent-core microcavity sensors employ a high-index quantum-dot coating in the channel of silica microcapillaries. Changes in the refractive index of fluids pumped into the capillary channel cause shifts in the microcavity fluorescence spectrum that can be used to analyze the channel medium.
JoVE Bioengineering
Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria
1Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, 2Clinical Research Laboratory, 81st Medical Group, Keesler Air Force Base
Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.
JoVE Bioengineering
Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents
Department of Biological Engineering, University of Missouri
Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.
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
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
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
Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface
Biomedical Engineering Department, Georgia Institute of Technology
An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin αLβ2 and intercellular adhesion molecule-1 as interacting receptors and ligands.
JoVE General
Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction
Institute of Molecular Medicine and Genetics, Georgia Health Sciences University
To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.
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 Applied Physics
Dry Oxidation and Vacuum Annealing Treatments for Tuning the Wetting Properties of Carbon Nanotube Arrays
Graduate Aeronautical Laboratories, California Institute of Technology
This article describes a simple method to fabricate vertically aligned carbon nanotube arrays by CVD and to subsequently tune their wetting properties by exposing them to vacuum annealing or dry oxidation treatment.
JoVE General
Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy
1Department of Systems Biology and Translational Medicine, College of Medicine, Cardiovascular Research Institute, Texas A&M Health Science Center, 2Department of Biomedical Engineering, Texas A&M University
This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.
JoVE Application Notes
LookOut Mycoplasma Elimination Kit - ADVERTISEMENT
Product Management, Sigma-Aldrich
The LookOut Mycoplasma elimination kit combines biological agents that reliably and completely eliminate mycoplasma contamination with minimal cytotoxic effect on cells.
JoVE Bioengineering
Quantitative Locomotion Study of Freely Swimming Micro-organisms Using Laser Diffraction
1Physics & Astronomy Department, Vassar College, 2Biology Department, Vassar College
Microscopic organisms like the free-swimming nematode C. elegans, live and behave in a complex three-dimensional environment. We report on a novel approach that provides analysis of C. elegans using diffraction patterns. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through a cuvette.
JoVE Neuroscience
Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging
Department of Neuroscience, Medical University of South Carolina
A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.
JoVE Bioengineering
Microfluidic Mixers for Studying Protein Folding
1Department of Physics and Astronomy, Michigan State University, 2Department of Mechanical Engineering, Hong Kong University of Science and Technology, 3Center for Biophotonics, University of California, Davis
In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 μs. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).
JoVE Immunology and Infection
Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins
1Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, 2Research service, 151, Veterans Affairs Greater Los Angeles Healthcare System, 3Departments of Medicine, Urology at David Geffen School of Medicine and Department of Microbiology, Immunology and Molecular Gentics, University of California Los Angeles (UCLA), 4Division of Infectious Diseases, 111F, Veterans Affairs Greater Los Angeles Health Care System
An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.
JoVE Immunology and Infection
Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce
1Center for Meat Safety and Quality, Department of Animal Sciences, Colorado State University, 2Rapid Microbial Detection and Control Laboratory, Department of Food Science and Human Nutrition, Iowa State University
This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).
JoVE General
Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap
1Department of Molecular Biology, Lewis-Sigler Institute for Integrative Genomics, Princeton University, 2Department of Physics, Lewis-Sigler Institute for Integrative Genomics, Princeton University
We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.
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 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
Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification
Department of Physiology and Biophysics, University of California, Irvine (UCI)
Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically ≥ 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.
JoVE General
CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV
1Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women's Hospital, 2Massachusetts Institute of Technology
JoVE Neuroscience
Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze
Department of Neuroscience, Friedman Brain Institute, Mount Sinai School of Medicine
We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.
JoVE Immunology and Infection
A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells
1Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Tsukuba, Japan, 2Safety Research Team, National Institute of Animal Health, Tsukuba, Japan
A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.
JoVE Neuroscience
Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures
Section on Critical Brain Dynamics, National Institute of Mental Health
A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.
JoVE Neuroscience
Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons
1Institute for Cellmorphology and molecular Neurobiology, Group for Cellbiology, Ruhr-University Bochum, 2Institute for Clinical Neurobiology, University of Wuerzburg
An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.
JoVE Clinical and Translational Medicine
Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts
1CR-UK Stromal-Tumour Interaction Group, Paterson Institute for Cancer Research, University of Manchester, 2Atopy Research Center, Juntendo University
Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.
JoVE Immunology and Infection
Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue
1Neuroscience Program, Uniformed Services University, 2Department of Anatomy, Physiology, and Genetics, Uniformed Services University, 3Molecular and Cell Biology, Uniformed Services University
Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.
JoVE Clinical and Translational Medicine
Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression
1Department of Preventive Medicine, University of Southern California, 2Institute for Women's Health, University College London
We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.
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
Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes
Research and Development, GE Healthcare Bio-Sciences AB
A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan™ DIGE technology.
JoVE Applied Physics
Fabrication of Nano-engineered Transparent Conducting Oxides by Pulsed Laser Deposition
1Department of Energy and NEMAS - Center for NanoEngineered Materials and Surfaces, Politecnico di Milano, 2Center for Nano Science and Technology, Instituto Italiano di Tecnologia
We describe the experimental method to deposit nanostructured oxide thin films by nanosecond Pulsed Laser Deposition (PLD) in the presence of a background gas. By using this method Al-doped ZnO (AZO) films, from compact to hierarchically structured as nano-tree forests, can be deposited.
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 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 Bioengineering
Bacterial Immobilization for Imaging by Atomic Force Microscopy
1Biological and Nanoscale Systems Group, Biosciences Division, Oak Ridge National Laboratory, 2Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, 3Department of Surgery, Eastern Virginia Medical School, 4Center for Nanophase Materials Sciences Division, Oak Ridge National Laboratory
Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).
JoVE Bioengineering
Polymer Microarrays for High Throughput Discovery of Biomaterials
1Laboratory of Biophysics and Surface Analysis, University of Nottingham, 2School of Molecular Medical Sciences, University of Nottingham, 3David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology
A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.
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 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
Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi
Department of Bioproducts and Biosystems Engineering, University of Minnesota
This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.
JoVE General
Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica
Department of Pharmacology and The Stem Cell Institute, University of Minnesota Medical School
An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.
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