JoVE General
Primary Human Bronchial Epithelial Cells Grown from Explants
Medicine, Faculty of Health Sciences, McMaster University
Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.
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 Clinical and Translational Medicine
Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry
1Sanford-Burnham Medical Research Institute, 2Division of Dermatology, University of California, San Diego, 3VA San Diego Healthcare Center, 4Moores Cancer Center, University of California, San Diego
Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable.
JoVE General
Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain
Molecular, Cellular and Developmental Biology, University of Michigan
A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.
JoVE General
A Novel Ex vivo Culture Method for the Embryonic Mouse Heart
McAllister Heart Institute, University of North Carolina at Chapel Hill
Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.
JoVE Clinical and Translational Medicine
Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats
Medicine/Nephrology, Indiana University School of Medicine
A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.
JoVE General
Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations
1Department of Biology and Biochemistry, University of Bath, 2Randall Division of Cell and Molecular Biophysics, King's College London
Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.
JoVE General
Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging
1Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología-Universidad Nacional Autónoma de México, 2Math and Sciences Department, Edison State College
Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.
JoVE Bioengineering
Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model
1Department of Microbiology and Immunology, Tulane University Medical School, 2Physician/Scientist Program, Tulane University Medical School, 3Department of Molecular and Cellular Biology, Baylor College of Medicine
Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.
JoVE Neuroscience
Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices
Department of Physiology, School of Medicine, University of Saarland, Homburg, Germany
In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.
JoVE General
Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells
1Department of Medical Oncology, Dana-Farber Cancer Institute, 2Harvard Medical School, Boston, MA, 3Sheba Cancer Research Center, Chaim Sheba Medical Center, 4Department of Pathology, Brigham and Women's Hospital
The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.
JoVE General
Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology
1Business Development, Corning Life Science, 2Applications, Corning Life Science, 3Product Development, Corning Life Science
An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.
JoVE Clinical and Translational Medicine
An in vivo Assay to Test Blood Vessel Permeability
We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.
JoVE Bioengineering
Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3
This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.
JoVE General
Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures
Bioengineering, University of Illinois
Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.
JoVE Neuroscience
The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury
Department of Neurology and Neurological Sciences, Stanford University School of Medicine
The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.
JoVE Neuroscience
Assessment of Motor Balance and Coordination in Mice using the Balance Beam
Department of Biology, California Institute of Technology
Deficits in fine motor coordination can be assessed with the balance beam test. Performance on the beam is quantified by the speed at which the beam is traversed and the number of times the mouse slips on the beam.
JoVE Clinical and Translational Medicine
A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor
1International Collaboration on Repair Discoveries (ICORD), University of British Columbia, 2Department of Orthopaedics, University of British Columbia
A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.
JoVE Neuroscience
An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium
1Department of Biology, Massachusetts Institute of Technology, 2Whitehead Institute of Biomedical Research
We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.
JoVE Immunology and Infection
Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy
1Division of Infectious Diseases, Department of Medicine, Immunology Institute, Mount Sinai School of Medicine, 2NSF Center for Biophotonics, University of California, Davis, 3Structural and Computational Biology Unit, European Molecular Biology Laboratory
This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.
JoVE Neuroscience
Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons
Department of Molecular Pharmacology and Experimental Therapeutics, Loyola University Chicago
We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.
JoVE Immunology and Infection
Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant
Center for Cell and Gene Therapy, Baylor College of Medicine
A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.
JoVE General
Engineering Cell-permeable Protein
Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.
JoVE Neuroscience
Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection
1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2Department of Microbiology & Immunology, Medical University of South Carolina, 3National Institute on Deafness and Other Communication Disorders, National Institutes of Health
Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.
JoVE General
A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning
1MRC LMCB, University College London, 2Center for Computational and Integrative Biology, Massachusetts General Hospital
A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.
JoVE General
A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium
Biosciences, College of Life and Environmental Sciences, The University of Exeter
We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.
JoVE Clinical and Translational Medicine
Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model
1Department of Orthopaedic Sports Medicine, Klinikum rechts der Isar der Technischen Universität München, 2Department of Radiology, Klinikum rechts der Isar der Technischen Universität München, 3Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar der Technischen Universität München, 4Department of Radiology, Uniklinik Köln
An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.
JoVE General
Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells
Department of Cell and Molecular Biology, Northwestern University
This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.
JoVE General
Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels
1Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, 2Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, 3Department of Anesthesiology, Medicine and Physiology, David Geffen School of Medicine, University of California, Los Angeles
We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.
JoVE General
Introduction to Solid Supported Membrane Based Electrophysiology
1Department of Biophysical Chemistry, Max Planck Institute of Biophysics, 2Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt
Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.
JoVE Immunology and Infection
The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis
Division of Gastroenterology, BC Children's Hospital
Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.
JoVE Immunology and Infection
Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation
1Department of Microbiology & Immunology, Virginia Commonwealth University- Massey Cancer Center, 2Department of Internal Medicine, Virginia Commonwealth University- Massey Cancer Center, 3Department of Surgery, Virginia Commonwealth University- Massey Cancer Center
An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.
JoVE Bioengineering
Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy
Department of Molecular and Cellular Biology, University of California Davis
We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.
JoVE General
Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat
The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.
JoVE Neuroscience
Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations
Department of Experimental Neurobiology, The Neurosciences Institute
We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.
JoVE Neuroscience
Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons
1Department of Neuroscience, Ohio State University College of Medicine, 2Department of Ophthalmology and Visual Science, University of Texas Medical School
An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.
JoVE Clinical and Translational Medicine
Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures
1Cardiology, Robotic Cardiac Electrophysiology and Arrhythmia Unit, La Paz University Hospital, 2Magnetecs Corp., 3Cardiology, Geffen School of Medicine at UCLA Los Angeles
This report provides a detailed description of a new remote navigation system based on magnetic driven forces, which has been recently introduced as a new robotic tool for human cardiac electrophysiology procedures.
JoVE General
Microcontact Printing of Proteins for Cell Biology
Department of Biomedical Engineering, Columbia University
Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.
JoVE General
Agarose Gel Electrophoresis for the Separation of DNA Fragments
Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles
A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.
JoVE General
Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles
1Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, 2Muscle Biology Research Group, University of Missouri-Kansas City, 3Pharmacology division, College of Pharmacy, DHLRI, Ohio State University
We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.
JoVE General
Plastic Embedding and Sectioning of Xenopus laevis Embryos
1Department of Developmental and Cell Biology, University of California, Irvine (UCI), 2University of California, Irvine (UCI)
Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.
JoVE General
Immunocytochemistry: Human Neural Stem Cells
Department of Pathology, University of California, Irvine (UCI)
Immunocytochemistry is a powerful method to determine the presence, subcellular localization, and relative abundance of an antigen of interest in cultured cells. This protocol presents an easy-to-follow series of steps that will enable one to conserve antibodies and get the most out of one's staining.
JoVE General
Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
1Department of Biological Sciences, University of Alberta, 2Department of Agriculture, Food and Nutrition Sciences, University of Alberta
An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.
JoVE Applied Physics
Encapsulation and Permeability Characteristics of Plasma Polymerized Hollow Particles
Department of Chemical Engineering, The Pennsylvania State University
We have used plasma enhanced chemical vapor deposition to deposit thin films ranging from a few nm to several 100 nm on nano-sized particles of various materials. We subsequently etch the core material to produce hollow nanoshells whose permeability is controlled by the thickness of the shell. We characterize the permeability of these coatings to small solutes and demonstrate that these barriers can provide sustained release of the core material over several days.
JoVE Clinical and Translational Medicine
Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots
1Department of Orthopaedic Sports Medicine, Klinikum rechts der Isar der Technischen Universität München, 2Department of Radiology, Klinikum rechts der Isar der Technischen Universität München, 3Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar der Technischen Universität München, 4Department of Radiology, Uniklinik Köln
An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.
JoVE Neuroscience
Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development
Department of Neuroscience and Physiology, SUNY Upstate Medical University
This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.
JoVE Neuroscience
A Novel Method for Assessing Proximal and Distal Forelimb Function in the Rat: the Irvine, Beatties and Bresnahan (IBB) Forelimb Scale
Department of Neurological Surgery, University of California, San Francisco
Here we will describe a rodent behavioral assay that can detect recovery of both proximal and distal forelimb function including digit movements during a naturally occurring behavior that does not require extensive training or deprivation to enhance motivation.
JoVE General
Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans
1Department of Biochemistry, University of Washington, 2Molecular and Cellular Biology Program, University of Washington
This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems
JoVE Immunology and Infection
Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus
1Center for Cell and Gene Therapy, Baylor College of Medicine, 2Pathology and Immunology, Baylor College of Medicine, 3Department of Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, 4Medicine, Baylor College of Medicine, 5Department of Pediatrics, Baylor College of Medicine
Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly naîve T cells.
JoVE Immunology and Infection
piggyBac Transposon System Modification of Primary Human T Cells
1Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, 2Department of Medicine, Division of Nephrology, Baylor College of Medicine, 3Department of Immunology and Pathology, Shinshu University School of Medicine, 4Center for Cell and Gene Therapy, Baylor College of Medicine, 5Department of Pediatrics, Baylor College of Medicine, 6Program in Cell and Molecular Biology, Baylor College of Medicine, 7Department of Molecular Virology and Microbiology, Baylor College of Medicine, 8Michael E. DeBakey VA Medical Center
We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.
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