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  JoVE Immunology and Infection

  
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  JoVE Clinical and Translational Medicine

  
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JoVE Science Education

General Laboratory Techniques

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Basic Methods in Cellular and Molecular Biology

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Model Organisms I

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Essentials of
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 JoVE Immunology and Infection

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

1Department of Pathology, Instituto Lauro de Souza Lima (ILSL), 2Laboratory Animal House, Instituto Lauro de Souza Lima (ILSL), 3Department of Microbiology, Instituto Lauro de Souza Lima (ILSL), 4Department of Pharmacology, Instituto Lauro de Souza Lima (ILSL)


JoVE 50620

Mycobacterium leprae, the causative agent of leprosy, does not grow in vitro. We describe an easy to follow protocol to prepare a bacillary suspension to ensure the maintenance of large quantities of M. leprae for a variety of applications. Protocols for propagation by mouse footpad inoculation, evaluation of viability, freezing and thawing bacillary stock are described in detail.

 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

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

1NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health


JoVE 51519

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

 JoVE Biology

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

1GIBCO, Life Technologies


JoVE 2237

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

 JoVE Neuroscience

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

1Department of Neurobiology and Behavior, University of California, Irvine


JoVE 2384

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

 JoVE Clinical and Translational Medicine

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in Clinically Relevant Animal Models and 3D Immunochemistry

1Department of Neurosurgery, Henry Ford Hospital


JoVE 51088

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

 JoVE Biology

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

1Department of Molecular and Medical Pharmacology, University of California, Los Angeles (UCLA), 2Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles (UCLA)


JoVE 52158

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

 JoVE Biology

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

1Animal Science Department, Iowa State University


JoVE 2764

Preimplantation embryos may be cryopreserved after placement into a hypertonic cryoprotective solution to cause cellular dehydration. After equilibration, ice crystal formation is induced in the solution surrounding the embryo. Further dehydration occurs as the embryo is slowly cooled to subzero temperatures before plunging into liquid nitrogen for storage.

 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 Biology

Freezing and Thawing Human Embryonic Stem Cells

1Research and Development, Stemgent


JoVE 1555

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

 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 Immunology and Infection

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

1Department of Clinical Neurosciences, John Van Geest Centre for Brain Repair, University of Cambridge, UK, 2NIHR Biomedical Research Center, University of Cambridge, UK


JoVE 51154

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

 JoVE Biology

Mouse Sperm Cryopreservation and Recovery using the I·Cryo Kit

1Genetically Engineered Models and Services, Charles River, 2Research Models and Services, Charles River


JoVE 3713

Here we demonstrate the newly developed I•Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I•Cryo kit and later successfully recovered by IVF.

 JoVE Biology

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

1Department of Anatomy and Developmental Biology, Monash University, 2Australian Regenerative Medicine Institute, Monash University


JoVE 51728

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

 JoVE Biology

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

1Department of Medicine, Albert Einstein College of Medicine, 2Department of Cell Biology, Albert Einstein College of Medicine


JoVE 52026

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

 JoVE Biology

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

1Molecular and Cellular Biology, University of California, Davis, 2Division of Basic Science, Fred Hutchinson Cancer Research Center - FHCRC


JoVE 1395

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

 JoVE Neuroscience

Isolation and Culture of Human Fungiform Taste Papillae Cells

1Monell Chemical Senses Center, 2New York University College of Dentistry, 3AFB International


JoVE 3730

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

 JoVE Neuroscience

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

1Department of Neurology, Beth Israel Deaconess Medical Center, 2Department of Obstetrics and Gynecology, Brigham and Women's Hospital, 3Department of Pathology, Beth Israel Deaconess Medical Center, 4Department of Pathology, Division of Neuropathology, Brigham and Women's Hospital


JoVE 2681

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

 JoVE Neuroscience

Derivation of Glial Restricted Precursors from E13 mice

1Hugo W. Moser Research Institute at Kennedy Krieger, Johns Hopkins University, 2Department of Neurology, Johns Hopkins School of Medicine, 3University of Maryland, 4Experimental Neurology, Biogen Idec, 5The Brain Science Institute, Johns Hopkins School of Medicine, 6Department of Pediatrics, Johns Hopkins School of Medicine


JoVE 3462

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

 JoVE Clinical and Translational Medicine

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

1CR-UK Stromal-Tumour Interaction Group, Paterson Institute for Cancer Research, University of Manchester, 2Atopy Research Center, Juntendo University


JoVE 3201

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

 JoVE Bioengineering

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

1Department of Medical Microbiology, Immunology & Cell Biology, Southern Illinois University School of Medicine, 2Division of Orthopaedics and Rehabilitation, Department of Surgery, Southern Illinois University School of Medicine, 3Department of Electrical and Computer Engineering, Biomedical Engineering Program, Southern Illinois University Carbondale, 4University of Illinois at Springfield


JoVE 51597

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

 JoVE Immunology and Infection

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

1Department of Biomedical Sciences, Cornell University


JoVE 2598

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

 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 Immunology and Infection

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

1Department of Biological Sciences, Hunter College and Graduate Center, City University of New York, 2Sunnybrook Research Institute, Department of Immunology, University of Toronto


JoVE 52119

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

 JoVE Clinical and Translational Medicine

In vitro Organoid Culture of Primary Mouse Colon Tumors

1Department of Molecular & Integrative Physiology, University of Michigan, 2Department of Internal Medicine, Division of Gastroenterology, University of Michigan


JoVE 50210

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

 JoVE Neuroscience

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

1Division of Biology, California Institute of Technology, 2Solomon H. Snyder Department of Neuroscience, Neurology, and Ophthalamology, Johns Hopkins University School of Medicine, 3Department of Radiation Oncology & Molecular Radiation Sciences, Johns Hopkins University School of Medicine, 4Department of Radiation Oncology, University Of Washington Medical Center, 5Institute for Cell Engineering and High-Throughput Biology Center, Johns Hopkins University School of Medicine


JoVE 50716

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

 JoVE Biology

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

1Department of Biochemistry, University of California - Riverside


JoVE 780

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

 JoVE Neuroscience

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

1Klinik und Poliklinik für Anästhesiologie, University of Wurzburg


JoVE 4022

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

 JoVE Neuroscience

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

1Department of Psychiatry, Johns Hopkins University, 2Department of Psychiatry and Behavioral Sciences, Howard University, 3Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, 4Department of Psychiatry, Sheppard Pratt Hospital, 5Department of Psychiatry, Indiana University


JoVE 51853

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

 JoVE Biology

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

1Department of Biomedical Genetics, Center for Oral Biology, James P Wilmot Cancer Center, University of Rochester


JoVE 1964

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

 JoVE Biology

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

1Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology


JoVE 734

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

 JoVE Biology

Generation of Myospheres From hESCs by Epigenetic Reprogramming

1Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, 2IRCCS Fondazione Santa Lucia


JoVE 51243

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

 JoVE Immunology and Infection

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

1Department of Medicine, Division of Hematology-Oncology, David Geffen School of Medicine at UCLA, 2UCLA AIDS Institute, 3Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, 4Department of Medical and Molecular Pharmacology, David Geffen School of Medicine at UCLA, 5Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA


JoVE 4181

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

 JoVE Neuroscience

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

1Department of Anatomy and Cell Biology, Wayne State University School of Medicine, 2Department of Opthalmology, Wayne State University School of Medicine


JoVE 51017

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

 JoVE Biology

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

1Humanitas Clinical and Research Center, Italy, 2Institute of Genetic and Biomedical Research (IRGB), National Research Council (CNR)


JoVE 50429

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

 JoVE Immunology and Infection

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

1Stony Brook Children's Hospital, State University of New York at Stony Brook, 2Department of Pediatrics, State University of New York at Stony Brook, 3Department of Molecular Genetics, State University of New York at Stony Brook, 4Department of Microbiology, State University of New York at Stony Brook


JoVE 3321

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

 JoVE Immunology and Infection

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

1Division of Microbiology, Tulane National Primate Research Center, 2Division of Comparative Pathology, Tulane National Primate Research Center, 3Division of Veterinary Medicine, Tulane National Primate Research Center


JoVE 3154

Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.

 JoVE Bioengineering

Harmonic Nanoparticles for Regenerative Research

1Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, 2Physics Department, GAP-Biophotonics, University of Geneva, 3Laboratoire d'Optique Biomédicale (LOB), Faculté des Sciences et Techniques de l'Ingénieur, École Polytechnique Fédérale de Lausanne, 4Department of Clinical Medicine, School of Medicine, Trinity College Dublin, 5School of Medicine and CRANN, Trinity College Dublin, 6Nikon AG Instruments


JoVE 51333

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

 JoVE Bioengineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

1University of California, San Diego


JoVE 2109

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

 JoVE Immunology and Infection

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

1Department of Immunology, MD Anderson Cancer Center - University of Texas, 2Department of Medicine, University of Miami


JoVE 1743

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

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