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HeLa Cells: The first continuously cultured human malignant Cell line, derived from the cervical carcinoma of Henrietta Lacks. These cells are used for Virus cultivation and antitumor drug screening assays.
 JoVE Biology

Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells

1Diagenode S.A., 2Diagenode Inc.


JoVE 52150

Methods for mapping in vivo protein-DNA interactions are becoming crucial for every aspect of genomic research but they are laborious, costly, and time consuming. Here a commercially available robotic liquid handling system that automates chromatin immunoprecipitation for mapping in vivo protein-DNA interactions with limited amounts of cells is presented.

 JoVE Immunology and Infection

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

1Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, 2Département de Biologie du Développement et des Cellules Souches, Institut Pasteur, Unité Macrophages et Développement de l'Immunité


JoVE 51601

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

 JoVE Immunology and Infection

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

1Viral Populations and Pathogenesis lab and CNRS 3015, Institut Pasteur


JoVE 2953

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

 JoVE Immunology and Infection

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

1Unité des Interactions Bactéries Cellules, Pasteur Institute, 2INSERM U604, 3Institut National de la Recherche Agronomique (INRA), USC2020, 4Institute of Biochemistry, ETH Zürich, 5Focal Area Infection Biology, Biozentrum, University of Basel


JoVE 51043

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

 JoVE Immunology and Infection

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

1Division of Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research, 2Aaron Diamond AIDS Research Center, Laboratory of Retrovirology, The Rockefeller University, NY


JoVE 4029

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

 JoVE Immunology and Infection

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

1Dynamique des Interactions Hôte Pathogène, Institut Pasteur, Paris, France, 2Imagopole, Institut Pasteur, Paris, France, 3Pathogenomique Mycobacterienne Integrée, Institut Pasteur, Paris, France


JoVE 50116

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

 JoVE Biology

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


JoVE 50317

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 Biology

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

1Department of Cell Biology, Harvard Medical School


JoVE 4140

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

 JoVE Bioengineering

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

1Department of Structural Biology, University of Pittsburgh School of Medicine, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine


JoVE 50386

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

 JoVE Biology

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

1RNA Biology, New England Biolabs


JoVE 3702

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

 JoVE Bioengineering

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

1Centre for Vascular Research and Australian Centre for Nanomedicine, The University of New South Wales, 2School of Chemistry and Australian Centre for Nanomedicine, The University of New South Wales


JoVE 50310

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

 JoVE Biology

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

1Department of Infectious Diseases, The Scripps Research Institute, 2Department of Chemistry, City College of New York


JoVE 51309

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

 JoVE Biology

Test Samples for Optimizing STORM Super-Resolution Microscopy

1Analytical Science Division, National Physical Laboratory


JoVE 50579

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

 JoVE Bioengineering

Cell Co-culture Patterning Using Aqueous Two-phase Systems

1Department of Biomedical Engineering, University of Michigan, 2Department of Macromolecular Science and Engineering, University of Michigan


JoVE 50304

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

 JoVE Biology

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

1Promega Corporation, 2MS Bioworks LLC


JoVE 51553

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.  Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

 JoVE Immunology and Infection

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

1Department of Molecular Biology and Microbiology, University of Central Florida


JoVE 3686

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 Immunology and Infection

Murine Model of CD40-activation of B cells

1Laboratory for Tumor and Transplantation Immunology, Department I of Internal Medicine, University Hospital of Cologne


JoVE 1734

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

 JoVE Biology

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

1McFerrin Department of Chemical Engineering, Texas A&M University, 2Department of Biomedical Engineering, Texas A&M University


JoVE 1749

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

 JoVE Bioengineering

Permeabilization of Adhered Cells Using an Inert Gas Jet

1Chemical Engineering, McGill University, 2Montreal Heart Institute


JoVE 50612

This protocol describes a method for the temporary permeabilization of adherent cells using an inert gas jet. This technique facilitates the transfer of genetic material and biomolecules into adherent mammalian cells by the utilization of mechanical forces to disrupt the plasma membrane.

 JoVE Bioengineering

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

1School of Biological and Health Systems Engineering, Arizona State University


JoVE 4415

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

 JoVE Biology

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

1Medical Biochemistry and Molecular Biology, Saarland University, 2Experimental and Clinical Pharmacology and Toxicology, Saarland University


JoVE 2730

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

 JoVE Biology

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

1Orflo Technologies, 2University of Utah


JoVE 3842

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

 JoVE Biology

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

1Department of Oncology, University of Wisconsin - Madison


JoVE 4305

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

 JoVE Chemistry

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

1Institute for Complex Molecular Systems & Laboratory of Macromolecular and Organic Chemistry, Eindhoven University of Technology & NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, 2NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR & IIT@NEST, Center for Nanotechnology Innovation, 3NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, 4IIT@NEST, Center for Nanotechnology Innovation


JoVE 50545

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

 JoVE Immunology and Infection

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

1Laboratory of Pediatric Infectious Diseases, Department of Pediatrics, Radboud university medical center


JoVE 50766

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

 JoVE Application Notes

Abcam Quantitative Cleaved PARP-1 High-Throughput In-Cell ELISA (ICE) Assay - ADVERTISEMENT

1Abcam, plc


JoVE 4200

Quantitative measurement of cleaved PARP-1 in fixed adherent or suspension cells by high-throughput In-Cell ELISA for using infra-red Li-Cor imaging system.

 JoVE Bioengineering

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

1CYTOO Cell Architects, Grenoble, France, 2Centre Commun de Quantimétrie, Faculté de Médecine Rockefeller, Lyon, France


JoVE 2514

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

 JoVE Biology

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

1Department of Neuroscience and Cell Biology, University of Texas Medical Branch


JoVE 2579

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

 JoVE Biology

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

1Department of Cell Biology and Molecular Genetics, University of Maryland


JoVE 50531

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

 JoVE Biology

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

1Department of Experimental Oncology, European Institute of Oncology


JoVE 51220

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

 JoVE Biology

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

1Department of Molecular Biology, Medical Biochemistry, and Pathology, Faculty of Medicine, Laval University, 2CHU de Quebec Research Center


JoVE 51164

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

 JoVE Immunology and Infection

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

1Department of Microbiology, Boston University School of Medicine


JoVE 51522

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

 JoVE Immunology and Infection

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

1Department of Virology, Universitätsklinikum Freiburg


JoVE 4028

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

 JoVE Biology

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


JoVE 2468

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 Clinical and Translational Medicine

Quantitative Analysis of Chromatin Proteomes in Disease

1Department of Anesthesiology, David Geffen School of Medicine at UCLA, 2Department of Medicine, David Geffen School of Medicine at UCLA, 3Department of Physiology, David Geffen School of Medicine at UCLA, 4Department of Internal Medicine, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah


JoVE 4294

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

 JoVE Biology

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2

1Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology


JoVE 1169

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

 JoVE Biology

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 1

1Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology


JoVE 1168

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

 JoVE Biology

Rapid Analysis and Exploration of Fluorescence Microscopy Images

1Green Center for Systems Biology, UT Southwestern Medical Center, 2Advanced Imaging Research Center, UT Southwestern Medical Center, 3Princeton University


JoVE 51280

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

 JoVE Bioengineering

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

1Department of Chemical Engineering, Massachusetts Institute of Technology, 2David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology


JoVE 50980

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

 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


JoVE 2925

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 Biology

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 3

1Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology


JoVE 1170

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

 JoVE Biology

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

1Department of Cellular and Molecular Medicine, Laboratory of Molecular and Cellular Signaling, KU Leuven


JoVE 50443

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

 JoVE Biology

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


JoVE 4002

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 Biology

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

1Bio-Rad Laboratories


JoVE 50948

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

 JoVE Clinical and Translational Medicine

Cholesterol Efflux Assay

1Baker IDI Heart and Diabetes Institute


JoVE 3810

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 Clinical and Translational Medicine

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

1Advanced Cell Diagnostics, Inc.


JoVE 51426

The presence of high-risk HPV in head and neck tumor tissue is associated with favorable outcomes. The recently developed RNA in situ hybridization technique called RNAscope allows direct visualization of HPV E6/E7 mRNA in FFPE tissue sections.

 JoVE Biology

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

1Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, Rockefeller University, 2Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, 3Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 4Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 5Genomics Resource Center, Rockefeller University


JoVE 2034

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

 JoVE Biology

Chromatin Isolation by RNA Purification (ChIRP)

1Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine


JoVE 3912

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

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