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October, 2006
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Immunology and Infection
Developmental Biology
Cell Biology: The study of the structure, behavior, growth, reproduction, and pathology of cells; and the function and chemistry of cellular components.
 JoVE Biology

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

1Max von Pettenkofer Institute, 2Department of Medicine, University of Cambridge, 3Institute for Informatics, Ludwig-Maximilians-University Munich

JoVE 50195

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

 JoVE Bioengineering

Sealable Femtoliter Chamber Arrays for Cell-free Biology

1Bredesen Center, University of Tennessee, Knoxville, 2Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, 3Department of Materials Science and Engineering, University of Tennessee, Knoxville

JoVE 52616

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

 JoVE Biology

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

1Department of Biology, California Institute of Technology, 2Department of Bioengineering, California Institute of Technology, 3Synthetic Biology Center, Department of Bioengineering, Massachusetts Institute of Technology, 4School of Physics and Astronomy, University of Minnesota

JoVE 50762

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

 JoVE Biology

Viability Assays for Cells in Culture

1Division of Pharmaceutical Sciences, Mylan School of Pharmacy, Duquesne University

JoVE 50645

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

 JoVE Neuroscience

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

1Virus and Centromere Team, Centre de Génétique et Physiologie Moléculaire et Cellulaire, CNRS UMR 5534, 2Université de Lyon 1, 3Laboratoire d'excellence, LabEX DEVweCAN, 4Institut de Virologie Moléculaire et Structurale, CNRS UPR 3296, 5Centre de Recherche en Cancérologie de Lyon, INSERM U1052, CNRS UMR 5286

JoVE 51091

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

 JoVE Biology

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

1Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University

JoVE 52321

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

 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

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

1Department of Biological Sciences, The University of Memphis

JoVE 4234

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

 JoVE Biology

Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

1Center for Vascular Biology Research, Department of of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School

JoVE 51320

Nanopodia are thin but fragile membrane channels that extend up to 100 μm from a cell's leading front or trailing rear and sense the cellular environment. Direct fixation at 37 °C, gentle washing, and avoidance of organic solvents like ethanol, methanol, or acetone and of higher Triton X-100 concentrations are required to observe these cellular structures.

 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

Purification of Transcripts and Metabolites from Drosophila Heads

1Department of Neurology, McKnight Brain Institute, University of Florida, 2Department of Entomology and Nematology, University of Florida, 3Genetics Institute, Department of Molecular Genetics and Microbiology, University of Florida, 4McKnight Brain Institute, Department of Neuroscience, Genetics Institute, Center for Translational Research on Neurodegenerative Diseases, and Center for Movement Disorders and Neurorestoration, University of Florida

JoVE 50245

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

 JoVE Biology

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

1Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven and Leuven Institute for Neuroscience and Disease (LIND)

JoVE 50523

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

 JoVE Neuroscience

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

1Neurodevelopment Group, School of Biosciences, University of Birmingham

JoVE 50306

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

 JoVE Immunology and Infection

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

1Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, 2Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, 3Department of Microbiology and Immunology, SUNY Upstate Medical University, 4Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center

JoVE 4165

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

 JoVE Biology

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

1Cardiovascular Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Division of Cardiology, Sapienza University

JoVE 50289

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

 JoVE Medicine

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

1Department of Leukemia, MD Anderson Cancer Center, 2Wake Forest Baptist Medical Center, Institute for Regenerative Medicine

JoVE 50385

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

 JoVE Biology

Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

1Basic Medical Sciences, University of Arizona College of Medicine - Phoenix

JoVE 3868

A rotating cell culture system that allows epithelial cells to grow under physiological conditions resulting in 3-D cellular aggregate formation is described. The aggregates generated display in vivo-like characteristics not observed in conventional culture models and serve as a more accurate organotypic model system for a multitude of scientific investigations.

 JoVE Biology

Isolation and Physiological Analysis of Mouse Cardiomyocytes

1Department of Medicine, Vanderbilt University, 2Department of Cell and Developmental Biology, Vanderbilt University

JoVE 51109

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

 JoVE Biology

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

1W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, 2School of Life Sciences, Chinese University of Hong Kong, 3Center for Cell Dynamics, Department of Biological Chemistry, Johns Hopkins University School of Medicine

JoVE 51964

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

 JoVE Biology

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

1Chemistry Department, Elon University, 2Department of Neurobiology and Anatomy, Wake Forest School of Medicine, 3Neuroscience Graduate Program, Wake Forest School of Medicine, 4ALS Center Translational Science Unit, Wake Forest School of Medicine

JoVE 52076

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

 JoVE Biology

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

1Max-Planck-Institute for Molecular Biomedicine and Institute of Cell Biology, 2Department of Internal Medicine, Yale Cardiovascular Research Center and Section of Cardiovascular Medicine

JoVE 51666

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

 JoVE Biology

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

1Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535

JoVE 51197

Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.

 JoVE Biology

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

1Department of Molecular Genetics, The Ohio State University

JoVE 52030

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein’s fluorescence intensity to that of an appropriate internal standard.

 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

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

1Department of Immunology and Infectious Diseases, Montana State University, 2Department of Molecular Biology, Princeton University

JoVE 50723

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

 JoVE Biology

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

1Department of Fundamental Microbiology, University of Lausanne

JoVE 50637

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

 JoVE Medicine

A Sensitive Method to Quantify Senescent Cancer Cells

1MILPAT (EA 4652), Université de Caen Basse-Normandie

JoVE 50494

Whether senescence prevents or promotes tumorigenesis remains controversial. Since chemotherapeutical drugs can induce cancer cells to senesce, studying senescence is essential for proposing new therapies. However, the standard and broadly used β-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

 JoVE Immunology and Infection

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

1Unité de Génétique, Papillomavirus et Cancer Humain (GPCH), Institut Pasteur, 2Cellule Pasteur, Université Sorbonne Paris Cité, 3Center for Cancer Systems Biology (CCSB), Harvard Medical School, Department of Cancer Biology, Dana Farber Cancer Institute

JoVE 50404

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

 JoVE Bioengineering

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

1Department of Pathology and Cell Biology, Columbia University, 2Herbert Irving Comprehensive Cancer Center, Columbia University

JoVE 50633

We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells.

 JoVE Biology

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

1Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute and Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 2Montreal Neurological Institute, 3Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute and Department of Pediatrics, University of Ottawa

JoVE 52295

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

 JoVE Biology

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

1Department of Biological Sciences, University of Notre Dame

JoVE 51644

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

 JoVE Neuroscience

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

1Department of Physical Medicine and Rehabilitation, VA Greater Los Angeles Healthcare System, 2Department of Pharmacology and Physiology, Institute for Neuroscience, The George Washington University School of Medicine and Health Sciences, 3Division of Genetics, Department of Medicine, Boston Children's Hospital, 4Howard Hughes Medical Institute, Boston Children's Hospital, 5Department of Pathology, Boston Children's Hospital, Harvard Medical School

JoVE 50333

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

 JoVE Bioengineering

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

1Cardiovascular Research Center, Massachusetts General Hospital and Harvard Medical School, 2Harvard Stem Cell Institute

JoVE 50288

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

 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

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

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

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

1Watson School of Biological Sciences, 2Cold Spring Harbor Laboratory, 3Departments of Medical Genetics, University of Oslo and Oslo University Hospital

JoVE 50088

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

 JoVE Bioengineering

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

1Section of Respiratory, Critical Care and Sleep Medicine, Department of Medicine, University of Illinois, 2Institute for Medicine and Engineering, University of Pennsylvania

JoVE 3886

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

 JoVE Bioengineering

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

1Torrey Pines Institute for Molecular Studies, 2Cascade LifeSciences Inc.

JoVE 50959

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

 JoVE Biology

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

1Department of Dermatology, Institute for Regenerative Cures, University of California, Davis, 2Departamento de Biologia, Centro de Biologia Molecular e Ambiental, Universidade do Minho, 3Department of Neurology and Center for Neuroscience, University of California, Davis Imaging of Dementia and Aging Laboratory, 4Department of Ophthalmology, Institute for Regenerative Cures, University of California, Davis

JoVE 52782

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

 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 Biology

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

1Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University

JoVE 4119

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

 JoVE Biology

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

1Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología-Universidad Nacional Autónoma de México, 2Math and Sciences Department, Edison State College

JoVE 50344

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

 JoVE Biology

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

1Cytori Therapeutics Inc, 2Division of Cardiac Surgery, Department of Surgery, David Geffen School of Medicine at UCLA, 3Division of Plastic and Reconstructive Surgery, Department of Surgery, David Geffen School of Medicine at UCLA, 4Department of Orthopedic Surgery, David Geffen School of Medicine at UCLA, 5Regenerative Bioengineering and Repair Laboratory, David Geffen School of Medicine at UCLA

JoVE 50585

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

 JoVE Immunology and Infection

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

1Department of Microbiology and Immunology, The Geisel School of Medicine at Dartmouth

JoVE 50598

Here we report a method for using type I and type II Δku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

 JoVE Biology

A Method for Culturing Embryonic C. elegans Cells

1Department of Physiology and Biophysics, University of Miami

JoVE 50649

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

 JoVE Biology

Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

1Department of Medicine, Albert Einstein College of Medicine, 2Diabetes Research and Training Center, Albert Einstein College of Medicine, 3Department of Genetics, Albert Einstein College of Medicine

JoVE 50246

An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

 JoVE Biology

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

1Institute for Physiology, Pathophysiology, & Toxicology, Centre for Biomedical Research and Training (ZBAF), University of Witten/Herdecke, 2Institute for Immunology & Experimental Oncology, Centre for Biomedical Research and Training (ZBAF), University of Witten/Herdecke

JoVE 51857

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

 JoVE Biology

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

1Department of Physiology and Biophysics, Chicago Medical School

JoVE 52509

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

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