JoVE Bioengineering
Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles
1Department of Chemistry & Biochemistry, University of Colorado Boulder, 2Biofrontiers Institute, University of Colorado Boulder
This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.
JoVE Clinical and Translational Medicine
Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion
1Department of Physiology and Functional Genomics, University of Florida, 2Department of Neurosurgery, McKnight Brain Institute, University of Florida, 3Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.
JoVE General
Making Patch-pipettes and Sharp Electrodes with a Programmable Puller
1Department of Molecular and Cellular Physiology, Stanford University, 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine
This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.
JoVE Immunology and Infection
Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics
Department of Physiology, School of Medicine, Louisiana State University Health Sciences Center
We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.
JoVE Neuroscience
Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells
1Department of Neurosurgery, University of Florida, 2Department of Anatomical Sciences, Shiraz University of Medical Sciences, 3STEMCELL Technologies, Inc.
This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.
JoVE Bioengineering
Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma
Department of Mechanical and Aerospace Engineering, The George Washington University
Anodic arc discharge is one of the most practical and efficient methods to synthesize various carbon nanostructures. To increase the arc controllability and flexibility, a non-uniform magnetic field was introduced to process the one-step synthesis of large-scale graphene flakes and high-purity single-walled carbon nanotubes.
JoVE General
Assessing Murine Resistance Artery Function Using Pressure Myography
In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGCα1-/- mice using pressure myography.
JoVE Bioengineering
Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry
We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.
JoVE Bioengineering
Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans
Department of Biology, University of Utah
Laser axotomy followed by time-lapse imaging is a sensitive way to assay the effects of mutations in C. elegans on axon regeneration. A high quality, but inexpensive, laser ablation system can be easily added to most microscopes. Time lapse imaging over 15 hours requires careful immobilization of the worm.
JoVE General
A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development
Department of Microbiology and Immunology, Loyola University Medical Center
We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.
JoVE Clinical and Translational Medicine
Murine Model of Wound Healing
1The Heart Research Institute, 2Sydney Medical School, University of Sydney, 3Cardiology Department, Royal Prince Alfred Hospital
A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.
JoVE General
Single Cell Electroporation in vivo within the Intact Developing Brain
1Brain Research Centre, University of British Columbia - UBC, 2Department of Cellular and Physiological Sciences, University of British Columbia - UBC
Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.
JoVE Applied Physics
Fabrication of Silica Ultra High Quality Factor Microresonators
1Department of Chemical Engineering and Materials Science, University of Southern California, 2Department of Electrical Engineering-Electrophysics, University of Southern California
We describe the use of a carbon dioxide laser reflow technique to fabricate silica resonant cavities, including free-standing microspheres and on-chip microtoroids. The reflow method removes surface imperfections, allowing long photon lifetimes within both devices. The resulting devices have ultra high quality factors, enabling applications ranging from telecommunications to biodetection.
JoVE Neuroscience
Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses
Department of Physiology & Biophysics, University of Colorado Medical Campus
Iontophoresis of neural agonists and antagonists during extracellular in vivo recordings is a powerful way to manipulate a neuron’s microenvironment. These manipulations can most easily be done via piggy-back multibarrel electrodes. Here we describe how to manufacture them and use them during auditory recordings.
JoVE Bioengineering
Mechanical Testing of Mouse Carotid Arteries: from Newborn to Adult
Department of Biomedical Engineering, Saint Louis University
Passive mechanical testing of mouse carotid arteries is described, with special consideration for adapting to different specimen ages. The procedures include determining the in vivo length of the artery, mounting it in a pressure myograph, recording data, measuring the unloaded dimensions and analyzing the resulting data.
JoVE General
Pressure-polishing Pipettes for Improved Patch-clamp Recording
Department of Molecular and Cellular Physiology, Stanford University School of Medicine
This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.
JoVE Bioengineering
Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness
1Section of Respiratory, Critical Care and Sleep Medicine, Department of Medicine, University of Illinois, 2Institute for Medicine and Engineering, University of Pennsylvania
Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.
JoVE Chemistry
LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations
1Institute of Biochemistry, Food Science, and Nutrition , The Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, 2Department of Physics and Astronomy, Ohio University
Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.
JoVE General
Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model
1Department of Cardiothoracic Surgery, Stanford University School of Medicine, 2Stanford University School of Medicine
This video demonstrates how to use a preclinical inexpensive and reliable model to study pathobiological and pathophysiological processes of in-stent restenosis development. Longitudinal in vivo monitoring using OCT (Optical Coherence Tomography) and analysis of OCT images are also demonstrated.
JoVE Bioengineering
Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study
1Department of Biomedical Engineering, Virginia Commonwealth University, 2Department of Anatomy and Neurobiology, Virginia Commonwealth University, 3Department of Cardiovascular Surgery, University Hospital of Geneva
The aim of this study was to mimic the native three layered architecture of the arterial wall. To accomplish this, electrospinning was employed with the use of a 3-1 (input-output) nozzle and blends of polycaprolactone, elastin, and collagen.
JoVE Clinical and Translational Medicine
Creation of Reversible Cholestatic Rat Model
Department of General Surgery, Providence Hospital and Medical Centers
Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Creation of a reversible cholestatic rat model can be challenging in view of the smaller size and unique hepatopancreatobiliary anatomy in rats. This video article demonstrates the creation of a reversible cholestatic model.
JoVE Applied Physics
In-situ Tapering of Chalcogenide Fiber for Mid-infrared Supercontinuum Generation
Edward L. Ginzton Laboratory, Stanford University
We describe a method for in-situ tapering of As2S3 fibers to achieve efficient mid-infrared supercontinuum generation. By tapering while monitoring the supercontinuum’s spectrum, the spectral width can be maximized for a fiber taper. In-situ fiber tapering can be applied to optimize the performance of other fiber-based devices.
JoVE General
Semi-automated Optical Heartbeat Analysis of Small Hearts
1Development and Aging Program, The Sanford Burnham Institute for Medical Research, 2Cardiac Electrophysiology Group, Dept. of Physiology, Anatomy and Genetics, The Sanford Burnham Institute for Medical Research, 3Biology Department and Heart Institute, San Diego State University
We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.
JoVE Applied Physics
Fabricating Metamaterials Using the Fiber Drawing Method
Institute of Photonics and Optical Sciences (IPOS), School of Physics, University of Sydney
Metamaterials at terahertz frequencies offer unique opportunities, but are challenging to fabricate in bulk. We adapt the fabrication procedure for microstructured polymer optical fibers to inexpensively fabricate metamaterials potentially on an industrial scale. We produce polymethylmethacrylate fibers containing ~10 μm diameter indium wires separated by ~100 μm, which exhibit a terahertz plasmonic response.
JoVE General
Using the optokinetic response to study visual function of zebrafish
Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.
JoVE Immunology and Infection
Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells
1Department of Systems Biology, Danish Technical University, 2Department of Biology, University of Copenhagen
Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).
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 Applied Physics
Construction and Testing of Coin Cells of Lithium Ion Batteries
1School of Materials Science and Engineering, Clemson University, 2Center for Optical Materials Science and Engineering Technologies, Clemson University
A protocol to construct and test coin cells of lithium ion batteries is described. The specific procedures of making a working electrode, preparing a counter electrode, assembling a cell inside a glovebox and testing the cell are presented.
JoVE Bioengineering
Elastomeric PGS Scaffolds in Arterial Tissue Engineering
1Department of Bioengineering, University of Pittsburgh, 2McGowan Institute for Regenerative Medicine, University of Pittsburgh
Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.
JoVE Bioengineering
Monitoring Protein Adsorption with Solid-state Nanopores
Department of Physics, Syracuse University
A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.
JoVE General
Fabrication of Amperometric Electrodes
1Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, 2Yale University School of Medicine
This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.
JoVE Neuroscience
Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window
1Bioengineering, University of California, San Diego, 2La Jolla Bioengineering Institute
Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.
JoVE Neuroscience
Measuring Motor Coordination in Mice
Department of Experimental Psychology, University of Oxford
Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.
JoVE Neuroscience
Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants
1 Neuroscience Lab/ Fac. Psicologia, University of Colima, 2Department of Neurosurgery, Johns Hopkins University
In this article, we show a method to make glass capillary needles with a 50-μm lumen. This technique significantly reduces the brain damage, minimizes passive diffusion of drugs and allows a precise targeting into the rodent brain.
JoVE Neuroscience
Organotypic Culture of Full-thickness Adult Porcine Retina
1Neurology and Neurosciences, University of Medicine and Dentistry of New Jersey - UMDNJ, 2Institute of Ophthalmology and Visual Science, University of Medicine and Dentistry of New Jersey - UMDNJ
Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.
JoVE Clinical and Translational Medicine
Isolation of Human Islets from Partially Pancreatectomized Patients
1Department of GI-, Thorax- and Vascular Surgery, University Hospital Carl Gustav Carus, University of Technology Dresden, 2Molecular Diabetology, Paul Langerhans Institute Dresden, 3Department of Pathology, University Hospital Carl Gustav Carus, University of Technology Dresden
The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.
JoVE Clinical and Translational Medicine
Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis
1University Heart Center Hamburg, TSI-Lab, Germany, 2Cardiovascular Research Center, University of Hamburg, 3Department of Medicine, Cardiology Division, Pulmonary Hypertension Program, University of Alberta, 4Department of Medicine, Stanford University School of Medicine, 5Department of Biomedical Sciences, Institute of Physiology, Pathophysiology, and Biophysics, University of Veterinary Medicine, Vienna, 6Translumina GmbH, Hechingen, 7Department of Cardiothoracic Surgery, Stanford University School of Medicine
This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.
JoVE General
Gene Transfer into Older Chicken Embryos by ex ovo Electroporation
1Albrecht-Kossel-Institute for Neuroregeneration, School of Medicine University of Rostock, 2Institute of Anatomy I, School of Medicine University of Jena
A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.
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 Applied Physics
Development of Whispering Gallery Mode Polymeric Micro-optical Electric Field Sensors
Mechanical Engineering Department, Southern Methodist University
A high-sensitivity photonic micro sensor was developed for electric field detection. The sensor exploits the optical modes of a dielectric sphere. Changes in the external electric field perturb the sphere morphology leading to shifts in its optical modes. The electric field strength is measured by monitoring these optical shifts.
JoVE General
In situ Quantification of Pancreatic Beta-cell Mass in Mice
Department of Medicine, University of Chicago
The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.
JoVE Neuroscience
Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation
1Neurosciences Graduate Program, University of Southern California, 2Zilkha Neurogenetic Institute, Department of Physiology and Biophysics, University of Southern California Keck School of Medicine
Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.
JoVE General
An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications
1Department of Biomedical Engineering, Cornell University, 2Current Address: Mechanical Engineering Department, Dogus University
In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.
JoVE Neuroscience
Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes
We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.
JoVE General
The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity
An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.
JoVE Clinical and Translational Medicine
In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer
Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado School of Medicine
The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.
JoVE Bioengineering
Use of Human Perivascular Stem Cells for Bone Regeneration
1Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, UCLA, 2UCLA and Orthopaedic Hospital, Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, UCLA, 3Department of Bioengineering, UCLA, 4Center for Cardiovascular Science, University of Edinburgh
Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.
JoVE Neuroscience
Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection
Lab for Molecular Mechanisms of Thalamus Development, RIKEN Brain Science Institute
A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.
JoVE Bioengineering
Silk Film Culture System for in vitro Analysis and Biomaterial Design
1Margaret M. Dyson Vision Research Institute, Weill Cornell Medical College, 2Department of Biomedical Engineering, Tufts University
Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.
JoVE General
Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.
As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns.
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