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  JoVE Biology

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October, 2006
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 JoVE Immunology and Infection

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

1Inserm U1019 - CNRS UMR 8024, Institut Pasteur de Lille, Université de Lille

JoVE 51114

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host cell using automated confocal microscopy.

 JoVE Immunology and Infection

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

1Cell Biology of Bacterial Infections, CNRS, FRE3689, CPBS, Université Montpellier

JoVE 52851

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

 JoVE Medicine

High Content Screening in Neurodegenerative Diseases

1Department of Clinical Genetics, VU University Medical Center, 2Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam

JoVE 3452

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

 JoVE Biology

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

1Department of Biological Sciences, University of Notre Dame

JoVE 52063

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

 JoVE Biology

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

1Cancer Biology, UCL Cancer Institute

JoVE 51882

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

 JoVE Medicine

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

1Department of Chemical and Biological Engineering, Iowa State University, 2Department of Genetics, Development and Cell Biology, Iowa State University, 3Biology Program, Iowa State University

JoVE 52242

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

 JoVE Biology

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

1Department of Biology, San Diego State University

JoVE 52452

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

 JoVE Bioengineering

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

1NanoScience Technology Center, University of Central Florida

JoVE 51866

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

 JoVE Medicine

Real-time Cytotoxicity Assays in Human Whole Blood

1Research and Development, Adheren, Inc, 2Research and Development, Eureka Therapeutics

JoVE 51941

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

 JoVE Medicine

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

1Department of Cell Biology, UT Southwestern Medical Center

JoVE 50369

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

 JoVE Biology

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

1Bioscience Division, High Content Analysis Research and Development, Millipore Inc

JoVE 1173

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

 JoVE Immunology and Infection

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

1Institute for Toxicology and Genetics, Karlsruhe Institute of Technology, Karlsruhe, Germany, 2Institute for Applied Informatics, Karlsruhe Institute of Technology, Karlsruhe, Germany

JoVE 4203

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

 JoVE Biology

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

1MRC LMCB, University College London, 2Center for Computational and Integrative Biology, Massachusetts General Hospital

JoVE 4382

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

 JoVE Bioengineering

Glutamine Flux Imaging Using Genetically Encoded Sensors

1Virginia Tech

JoVE 51657

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

 JoVE Biology

Development of automated imaging and analysis for zebrafish chemical screens.

1Pharmacology and Chemical Biology, University of Pittsburgh Drug Discovery Institute, 2Department of Microbiology and Molecular Genetics, University of Pittsburgh, 3Department of Pharmaceutical Sciences, University of Pittsburgh, 4Department of Chemistry, University of Pittsburgh

JoVE 1900

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

 JoVE Immunology and Infection

A Protocol for Analyzing Hepatitis C Virus Replication

1Liver Program at Regenerative Medicine Institute, Department of Biomedical Sciences, Department of Surgery, Cedars-Sinai Medical Center, 2Department of Surgery, David Geffen School of Medicine at UCLA

JoVE 51362

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

 JoVE Neuroscience

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

1Department of Genetics, UMR_S 968, Institut de la Vision, 2Department of Visual Information, UMR_S 968, Institut de la Vision, 3Exploratory Team, UMR_S 968, Institut de la Vision, 4Sorbonne Universités, Paris 06, UMR_S 968, Institut de la Vision, 5INSERM, U968, Institut de la Vision, 6CNRS, UMR_7210, Institut de la Vision

JoVE 51954

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 µm) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

 JoVE Neuroscience

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

1Department of Molecular and Cellular Biology, University of Guelph

JoVE 3422

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

 JoVE 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

JoVE 3525

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.

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