JoVE Clinical and Translational Medicine
A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells
Lombardi Comprehensive Cancer Center, Georgetown University
This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.
JoVE Immunology and Infection
Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System
Department of Immunology, College of Medicine, Mayo Clinic
The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.
JoVE General
Counting Human Neural Stem Cells
Department of Pathology, University of California, Irvine (UCI)
Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.
JoVE Application Notes
Orflo Moxi - ADVERTISEMENT
Moxi Z is the only automated cell counter that combines the Coulter Principle typically used in high-end cell counters with a patented thin-film sensor technology to allow for highly accurate (> 95%) and repeatable particle counting and sizing for a broad range of cell types - from mammalian cells to cells as small as wine yeast and more. Since today's workflows demand accurate quality control of samples, determining cell counts precisely has a significant impact on outcomes and downstream costs.
JoVE Immunology and Infection
Competitive Homing Assays to Study Gut-tropic T Cell Migration
Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School
Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.
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
Transfecting Human Neural Stem Cells with the Amaxa Nucleofector
Department of Pathology, University of California, Irvine (UCI)
Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.
JoVE Immunology and Infection
Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes
Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.
JoVE Clinical and Translational Medicine
Experimental Metastasis Assay
1Department of Biomedical Genetics, University of Rochester Medical Center, 2Department of Dermatology, University of Rochester Medical Center
This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.
JoVE General
Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish
1Department of Molecular Pathology, Massachusetts General Hospital, Harvard Medical School, 2Department of Molecular Pathology, Massachusetts General Hospital Cancer Center, Harvard Stem Cell Institute
Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.
JoVE Bioengineering
Cell Co-culture Patterning Using Aqueous Two-phase Systems
1Department of Biomedical Engineering, University of Michigan, 2Department of Macromolecular Science and Engineering, University of Michigan
Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.
JoVE Clinical and Translational Medicine
Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer
1Department of Oncology, Georgetown University, 2Lombardi Comprehensive Cancer Center, Georgetown University, 3Stem Cell Dynamics, Helmholtz Zentrum München - German Research Center for Environmental Health, 4Department of Medicine, Georgetown University, 5Department of Nanobiomedical Science and WCU Research Center of Nanobiomedical Science, Dankook University
Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.
JoVE General
Preparation of Drosophila S2 cells for Light Microscopy
Department of Cell Biology and Anatomy, University of Arizona (UOA)
Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.
JoVE General
Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy
1Department of Biochemistry and Molecular Medicine, University of California, Davis, 2NSF Center for Biophotonics Science & Technology, University of California, Davis, 3University of Tromsø, 4Department of Surgery (Division of Surgical Oncology), University of California, Davis, 5UC Davis Comprehensive Cancer Center, University of California, Davis, 6Department of Biological Chemistry, University of California, Davis
Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.
JoVE General
Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping
Institute of Biomedical Sciences, Federal University of Rio De Janeiro-UFRJ
Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.
JoVE General
Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin
Department of Physiology, Louisiana State University Health Sciences Center
Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.
JoVE General
Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter
1Orflo Technologies, 2University of Utah
The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.
JoVE Immunology and Infection
Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection
Department of Microbiology, Immunology and Pathology, Colorado State University
Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.
JoVE Bioengineering
Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)
Hochschule Aalen, Institut für Angewandte Forschung
Topology of cell adhesion on a substrate is measured with nanometre precision by variable-angle total internal reflection fluorescence microscopy (VA-TIRFM).
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 General
Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats
Department of Physiology and Biophysics, University of California, Irvine (UCI)
Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.
JoVE General
Retro-orbital Injection in Adult Zebrafish
1Department of Hematology and Oncology, Children’s Hospital Boston, 2Harvard Stem Cell Institute, Harvard Medical School, 3Department of Medical Oncology, Dana Farber Cancer Institute
Here we show how to do retro-orbital injection in adult zebrafish.
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
Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles
Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University
Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.
JoVE Immunology and Infection
Enzyme-linked Immunospot Assay (ELISPOT): Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens
1Department of Medicine, Vanderbilt University School of Medicine, 2Department of Microbiology and Immunology, Vanderbilt University School of Medicine
Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.
JoVE Immunology and Infection
Depletion and Reconstitution of Macrophages in Mice
1Department of Graduate Studies, University of British Columbia, 2Department of Molecular Biology, Vrije Universiteit Amsterdam, 3Department of Pediatrics, University of British Columbia
Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.
JoVE General
Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins
1Department of Radiation Oncology, Virginia Commonwealth University, 2Department of Biochemistry & Molecular Biology, Virginia Commonwealth University, 3Department of Anatomy & Neurobiology, Virginia Commonwealth University, 4Massey Cancer Center, Virginia Commonwealth University
This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.
JoVE General
Scale-Up of Mammalian Cell Culture using a New Multilayered Flask
Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.
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 General
Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters
Department of Biomedical Engineering, Rutgers University
We demonstrate a dark-field microscopy method based on Gabor-like filtering to measure subcellular dynamics within single living cells. The technique is sensitive to alterations in the structure of organelles, such as mitochondrial fragmentation.
JoVE General
Aseptic Laboratory Techniques: Plating Methods
Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles
When working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. A variety of plating methods are routinely used to isolate, propagate, or enumerate bacteria and phage, all of which incorporate procedures that maintain the sterility of experimental materials.
JoVE General
Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes
1Department of Flow and Image Cytometry, Roswell Park Cancer Institute, 2Flow Cytometry & Cell Sorting Resource Laboratory, University of Pennsylvania, 3SciGro, Inc., 4Department of Pathology and Laboratory Medicine, University of Pennsylvania
Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.
JoVE Neuroscience
Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology
1Neural Regeneration Laboratory and Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 2Carleton Immersive Media Studio, Azrieli School of Architecture and Urbanism, Carleton University
Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.
JoVE General
Synthesis of an In vivo MRI-detectable Apoptosis Probe
1Division of Cardiovascular Medicine, Department of Medicine, Stanford University Medical Center, 2Division of Cardiology, Department of Medicine, University of California, San Francisco, 3San Francisco VAMC
Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.
JoVE General
Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia
1Division of Cardiovascular Medicine, Stanford University, 2Department of Radiology, Stanford University
The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.
JoVE Immunology and Infection
Following Cell-fate in E. coli After Infection by Phage Lambda
1Department of Physics, University of Illinois at Urbana-Champaign, 2Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, 3Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine
This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.
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 Immunology and Infection
Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry
Department of Chemistry, Stony Brook University
Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.
JoVE General
In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy
1Department of Physics, University of Wisconsin - Milwaukee, 2Department of Biological Sciences, University of Wisconsin - Milwaukee
By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of Förster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.
JoVE General
4D Imaging of Protein Aggregation in Live Cells
Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.
JoVE General
Blood Collection from the American Horseshoe Crab, Limulus Polyphemus
1Department of Molecular and Cell Biology, University of California, Davis, 2Marine Biological Laboratory - MBL- woods hole, 3Department of Biological Sciences, Hunter College of CUNY
The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and α2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.
JoVE General
Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells
Stem Cell Research Unit, Medical University of Graz, Austria
Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.
JoVE General
In vivo-like Organotypic Murine Retinal Wholemount Culture
Institute for Anatomy, Department of Neuroanatomy, University of Duisburg-Essen
This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.
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 Clinical and Translational Medicine
Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma
1Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, 2Sensory Neuroscience Research Center, West Virginia University, 3Departments of Otolaryngology and Physiology, Center for Neuroscience, West Virginia University
A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.
JoVE Editorial
January 2012: This Month in JoVE
Here are some highlights from the January 2012 Issue of Journal of Visualized Experiments (JoVE).
JoVE General
Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice
1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behaviour, University of California, Irvine (UCI)
Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.
JoVE General
Germ Cell Transplantation and Testis Tissue Xenografting in Mice
Department of Comparative Biology and Experimental Medicine, University of Calgary
Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.
JoVE General
Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)
1Institut National de la Santé et de la Recherche Médicale, UMR 631, Parc scientifique de Luminy, 2Centre National de la Recherche Scientifique, UMR 6102, Parc scientifique de Luminy, 3Centre d'Immunologie de Marseille-Luminy, Aix-Marseille University, 4École Centrale Marseille, Technopôle de Château-Gombert, 5Institut Fresnel, Aix-Marseille University, 6Centre National de la Recherche Scientifique, UMR 6133, Aix-Marseille University
Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.
JoVE Immunology and Infection
Parasite Induced Genetically Driven Autoimmune Chagas Heart Disease in the Chicken Model
Chagas Disease Multidisciplinary Research Laboratory, University of Brasilia
The inoculation of Trypanosoma cruzi in fertile eggs prior to incubation renders the parasite kDNA minicircle integration in embryo cells genome. Crossbreeding reveals the vertical transfer of the mutations to progeny. The kDNA integrates into coding regions at several chromosomes and the chickens die with an inflammatory autoimmune heart disease.
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