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 Clinical and Translational Medicine
Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model
Institute of Human Physiology and Clinical Experimental Research, Semmelweis University
We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.
JoVE Clinical and Translational Medicine
The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease
1German Center for Neurodegenerative Diseases, DZNE, 2Laboratory of Functional Neurogenomics, Department of Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, University of Tübingen
Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.
JoVE Immunology and Infection
Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry
1Immunology, University of Toronto, 2Arthritis and Immune Disorder Research Centre, University Health Network (UHN)
This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.
JoVE General
Super-resolution Imaging of the Bacterial Division Machinery
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine
We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.
JoVE 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 Bioengineering
Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix
University of California, Davis
Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.
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 General
Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum
Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.
JoVE General
Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts
Department of Molecular and Cellular Biology, University of Guelph
In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.
JoVE General
Photobleaching Assays (FRAP & FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells
We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.
JoVE Neuroscience
Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis
1Research Service, Veterans Administration Medical Center, Memphis, TN, 2Department of Neurology, University of Tennessee Health Science Center, Memphis, TN, 3Department of Anatomy/Neurobiology, University of Tennessee Health Science Center, Memphis, TN
A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells.
JoVE Clinical and Translational Medicine
MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression
1Department of Pharmacology, Wayne State University, 2Barbara Ann Karmanos Cancer Institute, Wayne State University
We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.
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 General
Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET
Department of Cell and Molecular Biology, Karolinska Institutet
FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.
JoVE General
Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)
1Centre de Recherche de l’Institut du Cerveau et de la Moelle Épinière, Hôpital de la Pitié-Salpêtrière, 2Institut des Sciences Moléculaires d'Orsay, Université Paris-Sud, 3Centre de Photonique Biomédicale du Centre Laser, Université Paris-Sud
A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.
JoVE General
In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum
Biology Research and Development , Caliper Life Sciences
Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.
JoVE General
In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces
Institute of Biophysics, Johannes Kepler Universitat Linz
This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.
JoVE Immunology and Infection
Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein
We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.
JoVE General
Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)
1Institute for Clinical Neurobiology, University of Wuerzburg, 2Department of Synapses - Circuits - Plasticity, Max Planck Institute of Neurobiology, Martinsried, 3Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians University of Munich
Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.
JoVE Immunology and Infection
Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells
1Department of Physiology, Dartmouth College, 2Department of Biology, Indiana University Purdue University Indianapolis
This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.
JoVE General
Measuring the Kinetics of mRNA Transcription in Single Living Cells
RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.
JoVE Immunology and Infection
Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells
1Department of Chemical and Biomolecular Engineering, University of Houston, 2Division of Pediatrics, Research Unit 907, University of Texas MD Anderson Cancer Center
We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.
JoVE General
ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells
The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.
JoVE General
Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells
1The Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa
An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.
JoVE Bioengineering
Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells
1Department of Physics, King's College London, 2Department of Chemistry, Imperial College London, 3PhotoBiotics Ltd
Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.
JoVE Bioengineering
Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells
1Department of Cell Biology, Center for Cell Dynamics, Johns Hopkins University, 2Graduate School of Pharmaceutical Sciences, University of Tokyo, 3Biomedical Engineering, Johns Hopkins University
A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.
JoVE General
FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability
1Biotechnology Research Institute, AUT University and KODE Biotech Ltd, 2Shemyakin Institute of Bioorganic Chemistry RAS, Moscow, Russia
Function-Spacer-Lipid (FSL) constructs allow the surface characteristics of living cells and virions to be modified without loss of vitality. The method requires only simple contact of an FSL construct solution with a cell/virion and spontaneous and stable surface incorporation occurs.
JoVE General
Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro
Department of Biochemistry and Cell Biology, State University of New York
Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.
JoVE Bioengineering
Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context
1The Beatson Institute for Cancer Research, University of Glasgow, 2Section of Dermatology, School of Medicine, University of Glasgow
A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.
JoVE General
FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors
Förster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.
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 Clinical and Translational Medicine
Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting
Stem Cell and Cancer Research Institute, McMaster University
The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.
JoVE Editorial
September 2012: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
This September in JoVE, researchers from the School of Medicine at the Free University of Berlin demonstrate a novel method for studying how stroke patients compensate for visual field defects. To do this, our authors make use of a driving simulator complete with brakes, a steering wheel, and turn signals. Using driving simulation software and sophisticated eye tracking, researchers can compare the gaze behavior of stroke patients as they navigate through virtual driving courses with varying degrees of complexity. Though posterior cerebral artery infarction can lead to similar visual deficits in patients, some are able to navigate through the driving courses by developing compensatory eye movements, while others crash into dangerous obstacles, like wild boars. Through the analysis of compensatory gaze behavior employed by patients, our authors see great potential for using driving simulation as a tool to rehabilitate stroke patients trying to overcome the blind spots in their visual fields.
JoVE General
Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging
Institute for Molecular Cell Biology, Universty of Saarland
Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.
JoVE General
Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library
The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.
JoVE Bioengineering
Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and and Induced Pluripotent Stem Cells (iPSCs)
1Department of Medicine (Hematology, Oncology, and Transplant), University of Minnesota, Minneapolis, 2Stem Cell Institute, University of Minnesota, Minneapolis
This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.
JoVE General
Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS
1Department of Biochemistry, Temple University, 2Department of Anesthesiology and Pain Medicine, University of Washington
An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.
JoVE General
Bimolecular Fluorescence Complementation
Department of Pharmacology, University of Illinois at Chicago
The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.
JoVE General
Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)
Department of Cell Biology, Scripps Institute
Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).
JoVE Editorial
October 2011: This Month in JoVE
Here are some highlights from the October 2011 Issue of Journal of Visualized Experiments (JoVE).
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 Editorial
December 2011: This Month in JoVE
Here are some highlights from the December 2011 Issue of Journal of Visualized Experiments (JoVE).
JoVE Bioengineering
Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy
1Department of Thoracic and Cardiovascular Surgery and Inter-University Centre for Medical Technology Stuttgart-Tübingen (IZST), Eberhard Karls University, Tübingen, 2Department of Cell and Tissue Engineering, Fraunhofer Institute of Interfacial Engineering and Biotechnology (IGB) Stuttgart, Germany, 3Department for Medical Interfacial Engineering (IGVT), University of Stuttgart, Germany, 4Institute of Tissue Engineering and Regenerative Medicine, Julius-Maximillians University, Würzburg, Germany
Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.
JoVE Bioengineering
Video-rate Scanning Confocal Microscopy and Microendoscopy
1Program in Biophysics, Harvard University, 2Division of Health Sciences and Technology, Harvard-MIT, 3Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School
The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.
JoVE General
In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells
Departments of Radiology and Medicine (Cardiology), Stanford University School of Medicine
With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.
JoVE General
Time-lapse Imaging of Mitosis After siRNA Transfection
1Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, 2Fluorescence Microscopy Core Facility, University of Utah
Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.
JoVE Bioengineering
Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow
1Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, 2Micro and Nanotechnology Lab, University of Illinois at Urbana-Champaign, 3Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, 4Bioengineering, University of Illinois at Urbana-Champaign
Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.
JoVE General
Preparing Individual Drosophila Egg Chambers for Live Imaging
Department of Biochemistry, University of Oxford
The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.
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.
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