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  JoVE Biology

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  JoVE Neuroscience

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  JoVE Immunology and Infection

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  JoVE Medicine

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  JoVE Bioengineering

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  JoVE Engineering

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  JoVE Chemistry

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  JoVE Developmental Biology


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 JoVE Chemistry

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

1X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, 2Department of Physics and Astronomy, Northwestern University

JoVE 52370

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

 JoVE Biology

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

1Department of Microbiology and Immunology and Department of Pathology, Queen's University, 2Ask Science Products Inc.

JoVE 51710

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

 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

Clonogenic Assay: Adherent Cells

1Epigenomic Medicine, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Department of Pathology, The University of Melbourne, 3Epigenetics in Human Health and Disease, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 4Department of Anatomy and Cellular Biology, The University of Melbourne

JoVE 2573

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

 JoVE Biology

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

1SwitchGear Genomics

JoVE 3343

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

 JoVE Immunology and Infection

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

1Department of Neurosurgery, UCLA David Geffen School of Medicine, 2Department of Molecular and Medical Pharmacology, UCLA David Geffen School of Medicine, 3Department of Medicine, UCLA David Geffen School of Medicine, 4Brain Research Institute, UCLA David Geffen School of Medicine, 5Jonsson Comprehensive Cancer Center, UCLA David Geffen School of Medicine

JoVE 52416

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

 JoVE Medicine

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

1Women's Malignancies Branch, National Cancer Institute

JoVE 52446

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

 JoVE Immunology and Infection

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

1School of Clinical and Experimental Medicine, University of Birmingham, 2College of Medical and Dental Sciences, University of Birmingham, 3School of Immunity and Infection, University of Birmingham

JoVE 52480

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

 JoVE Neuroscience

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

1Regenerative Medicine Institute, Cedars-Sinai Medical Center

JoVE 51219

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.  Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

 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 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

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

1Department of Pharmacology, University of Illinois at Chicago, 2Department of Anesthesiology, University of Illinois at Chicago

JoVE 50329

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

 JoVE Immunology and Infection

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

1Department of Pharmacology, University of Minnesota, 2Department of Surgery, University of Minnesota, 33M Corporate Research Laboratory

JoVE 52195

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

 JoVE Immunology and Infection

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

1Department of Medicine, New York University School of Medicine, 2Departments of Pathology, New York University School of Medicine

JoVE 51646

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

 JoVE Immunology and Infection

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

1NIHR Biomedical Research Unit, Centre for Liver Research, School of Immunity and Infection, University of Birmingham

JoVE 51330

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

 JoVE Biology

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

1Molecular Pharmacology and Pathology Program, Department of Pathology & Bosch Institute, University of Sydney, 2Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University

JoVE 51322

Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

 JoVE Developmental Biology

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

1Translational Neuroscience Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, National Institutes of Health

JoVE 52298

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

 JoVE Bioengineering

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

1UMR CNRS 5557 Ecologie Microbienne, Université Lyon 1, Université de Lyon, 2Département Biosciences, INSA de Lyon, Université de Lyon, 3INSERM U758, Ecole Normale Supérieure de Lyon, Université de Lyon, 4Laboratoire de Génie Civil et Ingénierie Environnementale, INSA de Lyon, Université de Lyon

JoVE 4176

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

 JoVE Neuroscience

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

1Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, 2Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, 3Division of Neuropathology, Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, 4Curriculum in Genetics and Molecular Biology, University of North Carolina School of Medicine, 5Biological and Biomedical Sciences Program, University of North Carolina School of Medicine, 6Department of Radiation Oncology, Emory University School of Medicine, 7Department of Neurology, Neurosciences Center, University of North Carolina School of Medicine

JoVE 51763

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

 JoVE Developmental Biology

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

1Centre of Human and Aerospace Physiological Sciences, King's College London, 2Wellcome Trust-Medical Research Council, Cambridge Stem Cell Institute

JoVE 52049

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

 JoVE Bioengineering

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

1Centre for Vascular Research, University of New South Wales, 2School of Medical Sciences, University of New South Wales

JoVE 52423

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

 JoVE Bioengineering

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

1Department of Pulmonary Diseases, Institute for Cardiovascular Research, VU University Medical Center, 2Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center

JoVE 51300

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

 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 Medicine

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

1Department of Cancer Imaging and Metabolism, H. Lee Moffitt Cancer Center and Research Institute

JoVE 53070

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

 JoVE Biology

Isolation of Myofibroblasts from Mouse and Human Esophagus

1Department of Medicine, Keck School of Medicine, University of Southern California

JoVE 52215

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express α-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

 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 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 Medicine

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

1Department of Oncology, Georgetown University, 2Lombardi Comprehensive Cancer Center, Georgetown University, 3Stem Cell Dynamics, Helmholtz Zentrum München - German Research Center for Environmental Health, 4Department of Medicine, Georgetown University, 5Department of Nanobiomedical Science and WCU Research Center of Nanobiomedical Science, Dankook University

JoVE 50198

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

 JoVE Bioengineering

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

1Department of Surgery, Duke University Medical Center, 2Department of Biomedical Engineering, Duke University, 3School of Medicine, University of Pennsylvania, 4Department of Medicine, Division of Cardiology, Duke University Medical Center

JoVE 3349

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

 JoVE Immunology and Infection

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

1Institute of Nutrition, Friedrich Schiller University Jena

JoVE 51960

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

 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 Immunology and Infection

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

1Institute of Pharmaceutical Science, King's College London

JoVE 52989

Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells.

 JoVE Immunology and Infection

Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

1Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, 2Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, 3UK Veterinary Laboratories Agency, 4Visual Services, National Centers for Animal Health, Animal and Plant Health Inspection Service, United States Department of Agriculture

JoVE 52833

Long-term cultured interferon-γ enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

 JoVE Application Notes

3D Tissue Engineered Systems for Regenerative Approaches, Drug Discovery, and Toxicity Screening - ADVERTISEMENT

JoVE 5517

In vitro mammalian cell culture has served as an invaluable tool in cell biology for several decades. Classically, monolayer cultures of adherent cells were grown on flat and rigid two-dimensional (2D) substrates, such as polystyrene or glass. However, many cells, when isolated from tissues and placed onto stiff planar 2D cell culture surfaces, such as tissue culture plastic, become progressively flatter, divide aberrantly, and lose their differentiated phenotype1,2. While these two-dimensional cell culture studies have played a pivotal role in furthering our understanding of many biological processes, they do not emulate in vivo conditions.

 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 Medicine

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

1Department of Plastic and Hand Surgery, University of Freiburg Medical Centre

JoVE 3973

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

 JoVE Neuroscience

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

1University Clinics for Child and Adolescent Psychiatry, University of Zürich

JoVE 51748

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

 JoVE Neuroscience

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

1Department of Molecular Physiology & Biophysics, University of Iowa Carver College of Medicine, 2Department of Psychiatry, University of Iowa Carver College of Medicine, 3EZ BioResearch LLC

JoVE 51879

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

 JoVE Biology

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

1Brigham and Women's Hospital / Harvard Medical School, Department of Medicine, Cardiovascular Division, 2Weill Institute for Cell and Molecular Biology & Department of Biomedical Engineering, Cornell University

JoVE 3087

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

 JoVE Immunology and Infection

Depletion and Reconstitution of Macrophages in Mice

1Department of Graduate Studies, University of British Columbia, 2Department of Molecular Biology, Vrije Universiteit Amsterdam, 3Department of Pediatrics, University of British Columbia

JoVE 4105

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

 JoVE Biology

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

1Department of Medicine, Hematology-Oncology, University of California, Los Angeles, 2Department of Biological Chemistry, University of California, Los Angeles

JoVE 2559

mStrawberry OP9 cells allow for complete evaluation of all ES-derived progeny from co-culture.

 JoVE Immunology and Infection

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

1Department of Hematology and Oncology, University Medical Center Göttingen, 2Microbial Interface Biology Group, Research Center Borstel

JoVE 51554

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

 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 Biology

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

1Harvard Medical School, 2Division of Hematology/Oncology, Boston Children's Hospital, 3Department of Pediatric Oncology, Dana-Farber Cancer Institute, 4Howard Hughes Medical Institute

JoVE 52118

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

 JoVE Biology

Isolation and Culture of Neonatal Mouse Cardiomyocytes

1Randall and Cardiovascular Divisions, King’s College London, 2Division of Cardiology, School of Medicine, University of California San Diego

JoVE 50154

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

 JoVE Bioengineering

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

1Department of Biological Engineering, Massachusetts Institute of Technology, 2Environmental Toxicology, Chulabhorn Graduate Institute, 3Department of Biomedical Engineering, University of Minnesota

JoVE 50607

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

 JoVE Bioengineering

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

1Department of Thoracic and Cardiovascular Surgery and Inter-University Centre for Medical Technology Stuttgart-Tübingen (IZST), Eberhard Karls University, Tübingen, 2Department of Cell and Tissue Engineering, Fraunhofer Institute of Interfacial Engineering and Biotechnology (IGB) Stuttgart, Germany, 3Department for Medical Interfacial Engineering (IGVT), University of Stuttgart, Germany, 4Institute of Tissue Engineering and Regenerative Medicine, Julius-Maximillians University, Würzburg, Germany

JoVE 3977

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

 JoVE Immunology and Infection

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

1LD MacLean Surgical Research Laboratories, Department of Surgery, McGill University

JoVE 52687

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

 JoVE Immunology and Infection

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

1Department of Pathology and Molecule Medicine, McMaster University

JoVE 50490

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

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