Cell Biology:
The study of the structure, behavior, growth, reproduction, and pathology of cells; and the function and chemistry of cellular components.
JoVE General
In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts
Department of Cell Biology, Emory University
Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.
JoVE General
Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons
1Department of Genetics and Development, Columbia University, 2Department of Pathology and Cell Biology, Columbia University, 3Department of Neuroscience, Columbia University, 4Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School
Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.
JoVE General
In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging
Division of Cardiovascular Medicine, Stanford University
In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.
JoVE General
Preparation of Highly Coupled Rat Heart Mitochondria
1Faculty of Life Sciences, University of Manchester, 2School of Biological Sciences, Queen's University Belfast
We describe а protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.
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 General
Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR
Department of Biological Sciences, Southwestern Oklahoma State University
Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.
JoVE General
High-throughput Purification of Affinity-tagged Recombinant Proteins
Department of Life Sciences, Imperial College London
We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.
JoVE General
Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass
Department of Pathology, University of Utah School of Medicine
We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.
JoVE General
Microcontact Printing of Proteins for Cell Biology
Department of Biomedical Engineering, Columbia University
Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.
JoVE General
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
Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay
Thermo Scientific Solaris qPCR Products
The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.
JoVE General
Analyzing Large Protein Complexes by Structural Mass Spectrometry
Department of Biological Chemistry, Weizmann Institute of Science
Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.
JoVE General
T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis
Department of Biological Chemistry, Weizmann Institute of Science
Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.
JoVE General
Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation
Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.
JoVE Application Notes
Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay - ADVERTISEMENT
Thermo Scientific Solaris qPCR Products
JoVE General
Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection
Department of Biochemistry, University of Toronto
Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.
JoVE General
iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution
1Laboratory of Molecular Biology, Medical Research Council - MRC, 2European Bioinformatics Institute, EMBL Heidelberg, 3Computer and Information Science, University of Ljubljana, 4Wellcome Trust Genome Campus, Wellcome Trust Sanger Institute
The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.
JoVE General
Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps
1The Davis Heart and Lung Research Institute, The Ohio State University, 2Department of Pharmacology, College of Medicine, The Ohio State University
Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.
JoVE General
Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip
1Molecular Microbiology and Immunology, University of Missouri, 2Department of Surgery, University of Missouri, 3Child Health, University of Missouri
A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.
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 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 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 Application Notes
Western Blotting Troubleshooting Guide by Cell Signaling Technology - ADVERTISEMENT
Cell Signaling Technology, Inc.
Here we provide an extensive western blot troubleshooting guide based on our many years of experience, offering solutions to save you valuable time and reagents.
JoVE Application Notes
Western Blotting Protocol - ADVERTISEMENT
Cell Signaling Technology, Inc.
Here we provide a comprehensive western blot protocol developed and used at Cell Signaling Technology that includes optimal reagents and support information to ensure optimal results.
JoVE Bioengineering
Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study
Department of Engineering Mechanics, University of Nebraska-Lincoln
A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate the molecular/cellular mechanisms of blast-induced traumatic brain injury.
JoVE General
Population Replacement Strategies for Controlling Vector Populations and the Use of Wolbachia pipientis for Genetic Drive
Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University
In this interview, Jason Rasgon explains the concept of genetic drive and the characteristics of an effective gene drive system. The use of the endosymbiotic bacterium, Wolbachia pipientis, as a means to spread genes through mosquito populations, is hypothesized.
JoVE General
Maintaining Wolbachia in Cell-free Medium
Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University
This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.
JoVE General
Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination
Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University
Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.
JoVE General
Protocol for Mosquito Rearing (A. gambiae)
Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University
This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.
JoVE General
Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination
Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University
Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.
JoVE General
Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes
1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI), 2Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI)
In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.
JoVE General
Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes
1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI), 2Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI)
Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.
JoVE General
Injection of dsRNA into Female A. aegypti Mosquitos
1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI), 2Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI)
Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.
JoVE General
In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy
1Department of Anesthesiology and Critical Care, Shriners Hospital for Children, Massachusetts General Hospital, and Harvard Medical School, 2Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo
A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.
JoVE General
Long-term Imaging Mammalian Cells using Wide-Field Microscopy
Dept. of Cell Biology, Harvard Medical School
JoVE General
Propagation of Human Embryonic Stem (ES) Cells
Center for Regenerative Medicine, MGH - Massachusetts General Hospital
JoVE General
ES Cell-derived Neuroepithelial Cell Cultures
McLean Hospital, Harvard Medical School
Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).
JoVE General
Freezing Human ES Cells
Department of Molecular and Cell Biology, Harvard
Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.
JoVE General
Passaging HuES Human Embryonic Stem Cell-lines with Trypsin
Department of Molecular and Cell Biology, Harvard
In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.
JoVE General
Monitoring Actin Disassembly with Time-lapse Microscopy
Dept. of Systems Biology, Harvard Medical School
JoVE General
Human ES cells: Starting Culture from Frozen Cells
Department of Molecular and Cell Biology, Harvard
Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.
JoVE General
Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)
JoVE General
Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)
Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI)
This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.
JoVE General
Christopher Hughes: An in vitro model for the Study of Angiogenesis (Interview)
Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI)
Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.
JoVE General
Tracheotomy: A Method for Transplantation of Stem Cells to the Lung
Dept. of Cell Biology, Harvard Medical School
Significant breakthroughs in stem cell identification are continuously being made. To translate these discoveries, however, novel methods for cellular delivery must be devised. Here I report that the airways provide a safe route for stem cell transplantation to the lungs.
JoVE General
Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells
Childrens Hospital, Harvard Stem Cell Institute, Harvard Medical School
This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.
JoVE General
Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis
Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI)
This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.
JoVE General
MALDI Sample Preparation: the Ultra Thin Layer Method
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University
This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).
JoVE General
Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling
Department of Ecology and Evolutionary Biology, University of California, Los Angeles
Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.
JoVE General
Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes
1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI), 2Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI)
The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.
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