JoVE Neuroscience
Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching
MRC Centre for Synaptic Plasticity, University of Bristol
This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.
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 General
A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment
1Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, 2Bio-Medical Engineering Department, NED University of Engineering and Technology
Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.
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 Neuroscience
Organotypic Hippocampal Slice Cultures
Department of Physiology and Biophysics, University of Washington School of Medicine
We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.
JoVE Immunology and Infection
Intravital Imaging of the Mouse Popliteal Lymph Node
1Department of Pediatrics, Case Western Reserve University, 2Department of Pediatrics, Pathology and Biomedical Engineering, Case Western Reserve University
Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.
JoVE Neuroscience
Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons
By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.
JoVE Neuroscience
Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)
The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.
JoVE Immunology and Infection
Non-invasive Imaging of Leukocyte Homing and Migration in vivo
1Department of Pathology and Immunology, Washington University in St. Louis, 2National Institute of Neurological Disorders and Stroke, NINDS, NIH - National Institute of Health
Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.
JoVE General
Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)
Department of Biochemistry and Molecular Biology, Michigan State University
Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.
JoVE General
Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation
University of Massachusetts Medical School
Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.
JoVE General
Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes
Department of Zoology, University of British Columbia - UBC
The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.
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 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 General
Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells
Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies
We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.
JoVE Immunology and Infection
Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model
Department of Surgery, The Ohio State University Medical Center
Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.
JoVE General
Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)
1Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, 2Department of Microbiology and Immunology, McGill University, 3Department of Medicine, Division of Experimental Medicine, McGill University
A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.
JoVE General
Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University
Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).
JoVE Immunology and Infection
Intravital Microscopy of the Inguinal Lymph Node
1Interdisciplinary Science, University of Northern British Columbia, 2Northern Medical Program, University of Northern British Columbia
A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.
JoVE General
Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System
1Department of Biology, University of Western Ontario, 2Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada
We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.
JoVE Immunology and Infection
Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry
1Department of Immunology, University of Colorado School of Medicine, 2Division of Cell Biology, Department of Pediatrics, National Jewish Health, 3Department of Microbiology, Immunology, and Pathology, Colorado State University, 4Department of Immunology, National Jewish Health
We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.
JoVE General
Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity
DOE Plant Research Laboratory, Michigan State University
A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.
JoVE General
Heterokaryon Technique for Analysis of Cell Type-specific Localization
Department of Chemistry and Biochemistry, Worcester Polytechnic Institute- WPI
A flexible and efficient method for the characterization of cell type-specific protein localization and nucleocytoplasmic shuttling is described. This heterokaryon approach uses fluorescently-labeled fusion proteins to image protein localizations after cell fusion. The protocol is amenable to steady-state localizations or more dynamic determinations based on live cell imaging.
JoVE Neuroscience
Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons
FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.
JoVE General
Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH
1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behavior, University of California, Irvine (UCI)
Here we demonstrate a method for inducing and recording the progress of a delayed type-hypersensitivity (DTH) reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the effector / memory T cell response.
JoVE General
Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy
Cellular and Molecular Physiology, Yale University School of Medicine
In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.
JoVE General
In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells
1Department of Biochemistry, School of Medical Sciences, University of Bristol, 2Division of Immunity and Infection, School of Medicine, University of Birmingham
In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.
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
Single-Molecule Imaging of Nuclear Transport
1Department of Biology, Bowling Green State University, 2Center for Photochemical Sciences, Bowling Green State University
Single molecule microscopy approch provided novel insights into nuclear transport.
JoVE General
Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems
Department of Physiology and Biophysics, Robert Wood Johnson Medical School
Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.
JoVE General
Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding
1College of Nursing, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 2College of Science and Engineering, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 3School of Medicine, University of Washington
An in vitro method for preparing functional glucocorticoid receptor (GR)•hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.
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 General
Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry
1Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, 2Macromolecular Therapeutics Development Facility, Albert Einstein College of Medicine, Yeshiva University, 3Developmental and Molecular Biology, Albert Einstein College of Medicine, Yeshiva University
A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.
JoVE Neuroscience
Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae
Epigenetics and Progenitor Cells Keystone, Fox Chase Cancer Center
In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae
JoVE Neuroscience
Analysis of Dendritic Spine Morphology in Cultured CNS Neurons
1Department of Physiology, Northwestern University Feinberg School of Medicine, 2Department of Psychiatry and Behavioral Sciences, Northwestern University Feinberg School of Medicine
Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.
JoVE General
Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells
We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.
JoVE Bioengineering
Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound
1School of Biochemistry, University of Bristol, 2Smith and Nephew
This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.
JoVE General
Imaging Cell Shape Change in Living Drosophila Embryos
1Program in Cell & Molecular Biology, Baylor College of Medicine (BCM), 2Verna & Marrs McLean Department of Biochemistry & Molecular Biology, Baylor College of Medicine (BCM)
Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.
JoVE Immunology and Infection
Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs
1Department of Biomedical Engineering, Vanderbilt University, 2Vanderbilt Institute for Nanoscale Science & Engineering, Vanderbilt University, 3Interdisciplinary Materials Science Program, Vanderbilt University, 4Monroe Carell Jr. Children's Hospital, Vanderbilt University Medical Center, 5Department of Chemical & Biomolecular Engineering, Vanderbilt University, 6Department of Cancer Biology, Vanderbilt University
A hemolysis assay can be used as a rapid, high-throughput screen of drug delivery systems' cytocompatibility and endosomolytic activity for intracellular cargo delivery. The assay measures the disruption of erythrocyte membranes as a function of environmental pH.
JoVE Neuroscience
A Caenorhabditis elegans Model System for Amylopathy Study
We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human Aβ42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.
JoVE Clinical and Translational Medicine
Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging
1Department of Medical Biophysics, University of Western Ontario, 2London Regional Cancer Program, London Health Science Centre, 3Department of Pathology, Vanderbilt University, 4Translational Prostate Cancer Research Group, London Health Science Centre
We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.
JoVE Immunology and Infection
Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy
1Institute for Molecular and Clinical Immunology, Otto-von-Guericke University Magdeburg, 2Department of Immunoregulation, Helmholtz Center for Infection Research
We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).
JoVE General
Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging
1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behaviour, University of California, Irvine (UCI)
Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.
JoVE General
Dendra2 Photoswitching through the Mammary Imaging Window
1Department of Anatomy and Structural Biology, Albert Einstein College of Medicine - Yeshiva University, 2Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine - Yeshiva University, 3Hubrecht Institute-KNAW and University Medical Center Utrecht
Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.
JoVE Neuroscience
Automated Quantification of Synaptic Fluorescence in C. elegans
Department of Biological Sciences, University of Toledo
The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.
JoVE General
Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay
Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine
We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of β-galactosidase.
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 Clinical and Translational Medicine
Cholesterol Efflux Assay
Baker IDI Heart and Diabetes Institute
The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.
JoVE Bioengineering
Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique
1Laboratory of Nano- and Translational Medicine, Department of Radiation Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, 2Carolina Center for Nanotechnology Excellence, University of North Carolina
This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.
JoVE Immunology and Infection
Murine Superficial Lymph Node Surgery
1Maisonneuve-Rosemont Hospital Research Center, 2Department of Microbiology and Immunology, University of Montreal, 3Department of Medicine, University of Montreal
To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.
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