JoVE General
DNA Methylation: Bisulphite Modification and Analysis
1Epigenetics Group, Cancer Research Program, Garvan Institute of Medical Research, 2St Vincent's Clinical School, University of NSW
The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.
JoVE Immunology and Infection
Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides
1Department of Medical Microbiology and Immunology, University of California, Davis, 2Département Génétique et Développement, Institut Cochin, Université Paris Descartes
Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.
JoVE Neuroscience
Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain
Max Planck Institute of Psychiatry
A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.
JoVE General
Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1
1Department of Biomedical Informatics, The Ohio State University, 2Departments of Molecular Immunology and Clinical Oncology, Tohoku University
We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.
JoVE Neuroscience
Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes
STED Microscopy of Synaptic Function, European Neuroscience Institute Göttingen
FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.
JoVE Immunology and Infection
Protein Misfolding Cyclic Amplification of Prions
1Department of Civil Engineering, University of Nebraska at Lincoln, 2Department of Medical Microbiology and Immunology, Creighton University
Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.
JoVE General
Single Oocyte Bisulfite Mutagenesis
1Department of Obstretrics & Gynaecology, Schulich School of Medicine and Dentistry, University of Western Ontario, 2Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, 3Children's Health Research Institute
Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.
JoVE Immunology and Infection
Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions
Department of Medical Microbiology and Immunology, University of Wisconsin
Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.
JoVE Bioengineering
A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments
1Department of Biotechnology, Delft University of Technology, 2Delft Center for Systems and Control, Delft University of Technology
A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via β-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.
JoVE General
Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells
Department of Biology, University of Rochester
This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.
JoVE Bioengineering
Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer
Department of Bioengineering, Clemson University
A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.
JoVE Editorial
JoVE 4th Issue
JoVE General
Single Cell Fate Mapping in Zebrafish
1Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 2Division of Hematology/Oncology, Cincinnati Children's Hospital Medical Center
A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.
JoVE General
Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc
Department of Zoology, University of British Columbia - UBC
Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.
JoVE General
Using an EEG-Based Brain-Computer Interface for Virtual Cursor Movement with BCI2000
1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Wadsworth Center, New York State Dept. of Health
In this video, we demonstrate the steps required to run a brain-computer interface experiment, including setting up the EEG cap, calibrating the system, and training the user to move a cursor in two dimensions using imagined movements.
JoVE General
Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method
1Chemistry Research and Development, Luminex Corporation, 2Global Marketing, Luminex Corporation
An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.
JoVE General
Label-free in situ Imaging of Lignification in Plant Cell Walls
1Energy Biosciences Institute, University of California, Berkeley, 2Molecular Foundry, Lawrence Berkeley National Laboratory, 3Physical Biosciences Division, Lawrence Berkeley National Laboratory
A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.
JoVE Chemistry
Preparation and Use of Samarium Diiodide (SmI2) in Organic Synthesis: The Mechanistic Role of HMPA and Ni(II) Salts in the Samarium Barbier Reaction
Department of Chemistry, Lehigh University
A straightforward procedure for the preparation of samarium diiodide (SmI2) in THF is described. The role of two main additives namely hexamethylphosphoramide (HMPA) and Ni(acac)2 in Sm mediated reactions is demonstrated in the Sm-Barbier reaction.
JoVE General
Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format
1Max Planck Institute for Molecular Biomedicine, 2Medical Faculty, University of Münster
Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.
JoVE General
Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System
1Stemgent, 2Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology
The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.
JoVE General
Preparation of Quality Inositol Pyrophosphates
Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.
JoVE Applied Physics
Construction and Testing of Coin Cells of Lithium Ion Batteries
1School of Materials Science and Engineering, Clemson University, 2Center for Optical Materials Science and Engineering Technologies, Clemson University
A protocol to construct and test coin cells of lithium ion batteries is described. The specific procedures of making a working electrode, preparing a counter electrode, assembling a cell inside a glovebox and testing the cell are presented.
JoVE General
Selective Capture of 5-hydroxymethylcytosine from Genomic DNA
1Department of Human Genetics, Emory University School of Medicine, 2Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago
Described is a two-step labeling process using β-glucosyltransferase (β-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.
JoVE General
Bioenergetic Profile Experiment using C2C12 Myoblast Cells
1Buck Institute for Age Research, Novato, CA, 2Department of Pathology, Center for Free Radical Biology, University of Alabama at Birmingham - UAB, 3Seahorse Bioscience, North Billerica, MA
A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.
JoVE Clinical and Translational Medicine
Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain
1Department of Pathology, National Prion Disease Pathology Surveillance Center, Case Western Reserve University School of Medicine, 2Department of Neurology, National Prion Disease Pathology Surveillance Center, Case Western Reserve University School of Medicine
A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.
JoVE General
A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example
In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.
JoVE Applied Physics
Concurrent Quantitative Conductivity and Mechanical Properties Measurements of Organic Photovoltaic Materials using AFM
1Center for Nanoscale Materials, Argonne National Laboratory, 2Institute for Molecular Engineering, University of Chicago
Organic photovoltaic (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale inhomogeneity of OPV materials affects performance of photovoltaic devices. In this paper, we describe a protocol for quantitative measurements of electrical and mechanical properties of OPV materials with sub-100 nm resolution.
JoVE General
Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards
Center for the Integrative Study of Animal Behaviour, Macquarie University
Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.
JoVE Clinical and Translational Medicine
Mouse Model of Middle Cerebral Artery Occlusion
1Department of Neurology, Ernest Gallo Clinic and Research Center, University of California, San Francisco, 2Department of Biological Sciences, Kent State University
We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.
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 Clinical and Translational Medicine
Modeling and Imaging 3-Dimensional Collective Cell Invasion
1Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, 2The Beatson Institute for Cancer Research
Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.
JoVE General
Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion
1Division of Gastroenterology, Hepatology & Nutrition, Department of Pediatrics, The Children's Hospital of Philadelphia, 2Department of Medicine, University of Pennsylvania
A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.
JoVE Bioengineering
Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy
1Center for Biophotonics Science and Technology, University of California, Davis, 2Department of Pathology and Laboratory Medicine, University of California, Davis
We discuss the construction and operation of a complex nonlinear optical system that uses ultrafast all-optical switching to isolate Raman from fluorescence signals. Using this system we are able to successfully separate Raman and fluorescence signals utilizing pulse energies and average powers that remain biologically safe.
JoVE General
Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay
We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.
JoVE General
Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells
1UCSF Diabetes Center and Department of Cell and Tissue Biology, University of California, San Francisco, 2Department of Biology, University of Copenhagen, Denmark, 3National Institute of Nutrition and Seafood Research, Bergen, Norway
Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.
JoVE General
Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software
Human Genetics, University of Michigan
The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.
JoVE General
Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test
Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Brasil
Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.
JoVE Immunology and Infection
Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes
Department of Immunology, Duke University
We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.
JoVE Applied Physics
Probing and Mapping Electrode Surfaces in Solid Oxide Fuel Cells
1Center for Innovative Fuel Cells and Battery Technologies, School of Materials Science and Engineering, Georgia Institute of Technology, 2School of Chemistry and Biochemistry, Georgia Institute of Technology
We present a unique platform for characterizing electrode surfaces in solid oxide fuel cells (SOFCs) that allows simultaneous performance of multiple characterization techniques (e.g. in situ Raman spectroscopy and scanning probe microscopy alongside electrochemical measurements). Complementary information from these analyses may help to advance toward a more profound understanding of electrode reaction and degradation mechanisms, providing insights into rational design of better materials for SOFCs.
JoVE General
Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy
1Department of Psychology, University of Michigan, Ann Arbor, 2Department of Psychology, University of Toronto Scarborough
Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.
JoVE General
Presynaptically Silent Synapses Studied with Light Microscopy
1Department of Psychiatry, Washington University School of Medicine, 2Department of Anatomy, Washington University School of Medicine, 3Department of Neurobiology, Washington University School of Medicine
Glutamatergic synapses can switch from an active mode to a silent mode. We demonstrate that presynaptic activity status in dissociated culture of rodent neurons is visualized using a fixable form of the FM1-43 dye to visualize active synapses and immunostaining with vGluT-1 antibody to visualize all glutamate synapses.
JoVE Neuroscience
Simultaneous fMRI and Electrophysiology in the Rodent Brain
1Biomedical Engineering, Emory University, 2Biomedical Engineering, Georgia Institute of Technology, 3Biology, Emory University
We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.
JoVE Immunology and Infection
The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation
Department of Immunology, John Curtin School of Medical Research, Australian National University
CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.
JoVE General
Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)
1Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California at Los Angeles, 2Crump Institute for Molecular Imaging, David Geffen School of Medicine, University of California at Los Angeles, 3California NanoSystems Institute, University of California at Los Angeles, 4Nuclear Medicine, PET Center, Shanghai Medical Collegea, Fudan University, 5Electronics and Information Engineering, College of Electronics and Information Engineering, Wuhan Textile University
A facile, one-pot synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) was developed based on a non-aqueous, three-step radiochemical process. Using microwave heating, the entire procedure can be completed in less than 30 min, or 60 min with further purification by preparative HPLC. The decay-corrected radiochemical yields (RCYs) were 35-5% (n > 30).
JoVE General
Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran
1Department of Biological Sciences, Vanderbilt University, 2Department of Chemical and Systems Biology, Stanford University
This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.
JoVE Immunology and Infection
Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)
Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.
JoVE Immunology and Infection
Estimating Virus Production Rates in Aquatic Systems
Department of Microbiology, University of Tennessee
The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.
JoVE Bioengineering
Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition
1Graduate Group in Biophysics, University of California San Francisco, 2Department of Biochemistry and Biophysics, University of California San Francisco
Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.
JoVE Bioengineering
Continuously-stirred Anaerobic Digester to Convert Organic Wastes into Biogas: System Setup and Basic Operation
Department of Biological and Environmental Engineering, Cornell University
Laboratory-scale anaerobic digesters allow scientists to research new ways of optimizing existing applications of anaerobic biotechnology and to evaluate the methane producing potential of various organic wastes. This article introduces a generalized model for the construction, inoculation, operation, and monitoring of a laboratory-scale continuously stirred anaerobic digester.
JoVE General
Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method
Molecular Biophysics and Biochemistry, Yale University
This article describes a standard method to get a three-dimensional (3D) reconstruction of biological macromolecules using negative staining electron microscopy (EM). In this protocol, we explain how to get the 3D structure of the Saccharomyces cerevisiae exosome complex at medium resolution using the random conical tilt reconstruction method (RCT).
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