JoVE Immunology and Infection
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)
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.…
Published March 23, 2014. Keywords: Infectious Diseases, Mycobacterium leprae, skin diseases, bacteria, maintenance, viability, freezing, athymic nude mice
1Regenerative Medicine Institute, Cedars-Sinai Medical Center
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.
Published June 15, 2014. Keywords: Neuroscience, neural progenitor cell, neural precursor cell, neural stem cell, passaging, neurosphere, chopping, stem cell, neuroscience, suspension culture, good manufacturing practice, GMP
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
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.
Published July 24, 2014. Keywords: Stem Cell Biology, Pluripotent stem cells, human embryonic stem cells, induced pluripotent stem cells, cell culture, non-colony type monolayer, single cell, plating efficiency, Rho-associated kinase, Y-27632, transfection, transduction
JoVE Immunology and Infection
1Department of Microbiology and Immunology, Wake Forest University Health Sciences, 2R&D Department, R.J. Reynolds Tobacco Company
Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure. …
Published January 10, 2015. Keywords: Immunology, Tobacco product preparation, whole smoke-conditioned medium, human peripheral blood mononuclear cells, PBMC, lipopolysaccharide, cell death, secreted cytokines, intracellular cytokines, K562 killing assay.
1Department of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, 2Department of Medicine, Section of Gastroenterology and Hepatology, Baylor College of Medicine
We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.
Published March 6, 2015. Keywords: Medicine, Intestinal stem cells, 3-dimensional cell culture, human, small intestine, colon, biopsy, enteroids, minigut, epithelial organoids, in-vitro, colonoids, enterospheres
JoVE Developmental Biology
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
A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.
Published January 28, 2015. Keywords: Developmental Biology, Hematopoietic progenitor cell, neural stem cell, blood, Sendai virus, neuron, differentiation
1GIBCO, Life Technologies
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.
Published July 15, 2010. Keywords: Cellular Biology, iPS, pluripotent, stem cells, cell culture, medium, media, feeder-free
JoVE Developmental Biology
1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Institute of Stem Cell Biology and Regenerative Medicine, Cardiovascular Medicine Division, Department of Medicine, Child Health Research Institute, Stanford University School of Medicine
Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.
Published March 18, 2015. Keywords: Developmental Biology, Human induced pluripotent stem cells, cardiac differentiation, small molecule compounds, cardiomyocytes, glucose starvation
JoVE Developmental Biology
1Institute for Biochemistry and Molecular Biology, Ulm University
Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.
Published June 25, 2015. Keywords: Developmental Biology, Tetracycline-controlled transcriptional activation, TetON, zebrafish, Regeneration, fin, tissue-specific gene expression, doxycycline, cryosectioning, transgenic, Tol2, I-SceI, anesthesia
JoVE Developmental Biology
1Center for Biomedicine, European Academy Bozen/Bolzano (EURAC), 2Laboratory of Medical Genetics, Fondazione IRCCS Ca´ Granda, Ospedale Maggiore Policlinico, 3Del E. Webb Center for Neuroscience, Aging & Stem Cell Research, Sanford-Burnham Medical Research Institute
Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.
Published June 5, 2015. Keywords: Developmental Biology, Stem cell biology, cellular biology, molecular biology, induced pluripotent stem cells, peripheral blood mononuclear cells, reprogramming, episomal plasmids.
1Department of Neurobiology and Behavior, University of California, Irvine
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.
Published November 11, 2010. Keywords: Neuroscience, cryopreservation, neuron, astrocyte, neuronal stem cells, tissue culture, brain cortex
1Department of Neurosurgery, Henry Ford Hospital
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.
Published January 7, 2014. Keywords: Medicine, Primary Cell Culture, animal models, Nervous System Diseases, Neoplasms, glioblastoma, neurosphere, surgical specimens, long-term self-renewal
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)
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.
Published November 26, 2014. Keywords: Stem Cell Biology, Human induced pluripotent stem cells, STEMCCA, factor-free, GMP, xeno-free, quantitative PCR
1Randall and Cardiovascular Divisions, King’s College London, 2Division of Cardiology, School of Medicine, University of California San Diego
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.…
Published September 6, 2013. Keywords: Cellular Biology, Biomedical Engineering, Bioengineering, Molecular Biology, Cell Culture Techniques, Primary Cell Culture, Cell Culture Techniques, Primary Cell Culture, Cell Culture Techniques, Primary Cell Culture, Cell Culture Techniques, Disease Models, Animal, Models, Cardiovascular, Cell Biology, neonatal mouse, cardiomyocytes, isolation, culture, primary cells, NMC, heart cells, animal model
1Animal Science Department, Iowa State University
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.
Published August 5, 2011. Keywords: Developmental Biology, embryo, cryopreservation, cattle, sheep, goats
1Research and Development, Stemgent
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.…
Published December 24, 2009. Keywords: Developmental Biology, Human embryonic stem cell, medium, hES, maintenance, thaw, ES cells, stem cell, cell culture, pluripotency, differentiation, passage, freeze
1Department of Biological Sciences, University of Notre Dame
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.
Published August 9, 2014. Keywords: Cellular Biology, zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
1Institute of Anatomy and Cell Biology, University of Würzburg, 2Institute of Reconstructive Neurobiology, University of Bonn, 3German Cancer Research Center, Heidelberg
Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.…
Published July 29, 2015. Keywords: Neuroscience, Direct conversion, lineage reprogramming, transgene-free reprogrammed cells, neural stem cells, transdifferentiation, neuronal differentiation, glial differentiation, stem cell biology, disease modeling, neural cell replacement, stem cell therapy.
JoVE Immunology and Infection
1Department of Clinical Neurosciences, John Van Geest Centre for Brain Repair, University of Cambridge, UK, 2NIHR Biomedical Research Center, University of Cambridge, UK
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.…
Published April 15, 2014. Keywords: Immunology, Somatic neural stem/precursor cells, neurodegenerative disorders, regenerative medicine, multiple sclerosis, experimental autoimmune encephalomyelitis, systemic delivery, intravenous, intracerebroventricular
1Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole polytechnique fédérale de Lausanne
We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.
Published January 8, 2015. Keywords: Medicine, Hormone signaling, breast cancer, reduction mammoplasty, breast tissue microstructures, ex vivo model, estrogen, progesterone, mammary epithelial cells, tissue digestion, paracrine signaling, microenvironment, tissue architecture
1Genetically Engineered Models and Services, Charles River, 2Research Models and Services, Charles River
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.…
Published December 12, 2011. Keywords: Basic Protocols, Cryopreservation, Sperm, In vitro fertilization (IVF), Mouse, Genetics
1Department of Medicine, Albert Einstein College of Medicine, 2Department of Cell Biology, Albert Einstein College of Medicine
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.
Published November 3, 2014. Keywords: Cancer Biology, Colorectal Cancer, Mouse, Small Intestine, Crypt, Organoid, Diet, Metabolism, Extracellular Acidification Rate, Oxygen Consumption Rate
1Department of Anatomy and Developmental Biology, Monash University, 2Australian Regenerative Medicine Institute, Monash University
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.
Published September 6, 2014. Keywords: Stem Cell Biology, Induced pluripotent stem cells; reprogramming; intermediates; fluorescent activated cells sorting; cell surface marker; reprogrammable mouse model; derivation of mouse embryonic fibroblasts
1Department of Cell Biology, UT Southwestern Medical Center, 2National Institute of Neurological Disorders and Stroke, National Institute of Health, 3Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 4Division of Genetics and Genomics, Boston Children's Hospital
This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.
Published January 18, 2015. Keywords: Medicine, Biopsy, skeletal muscle, skin, tissue dissociation, myoblast purification, myoblast immortalization, cell freezing
1Molecular and Cellular Biology, University of California, Davis, 2Division of Basic Science, Fred Hutchinson Cancer Research Center - FHCRC
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.
Published July 6, 2009. Keywords: Developmental Biology, Zebrafish, sperm, cryopreservation, TILLING
1Monell Chemical Senses Center, 2New York University College of Dentistry, 3AFB International
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.
Published May 17, 2012. Keywords: Neuroscience, Fungiform, taste cells, cell culture, gustducin, calcium imaging, molecular biology, human fungiform papillae
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
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.
Published May 25, 2011. Keywords: Neuroscience, neural stem cell, human, brain, cortex, fetus, cell culture
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
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.
Published June 20, 2012. Keywords: Neuroscience, Physiology, Medicine, periventricular leukomalacia, oligodendrocytes, glial restricted precursors, spinal cord, cell culture
1CR-UK Stromal-Tumour Interaction Group, Paterson Institute for Cancer Research, University of Manchester, 2Atopy Research Center, Juntendo University
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.…
Published October 25, 2011. Keywords: Medicine, cancer, stromal myofibroblasts, experimentally generated carcinoma-associated fibroblasts (exp-CAFs), fibroblast, human mammary carcinomas, tumour xenografts
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
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.
Published April 30, 2014. Keywords: Bioengineering, Anterior Cruciate Ligament, Tissue Engineering, hACL derived cells, PLAGA, in vitro expansion, ACL partial tears
1Department of Medicine, Medical College of Wisconsin
This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.
Published January 28, 2015. Keywords: Medicine, Stem cell biology, Urine derived Cells (UCs), Induced Pluripotent Stem Cells (iPSCs), reprogramming, Sendai Virus (SeV), viral transduction, iPSC-derived Cardiomyocytes (iCMs), regenerative medicine, Muscular Dystrophy (MD), dystrophic cardiomyopathy.
JoVE Immunology and Infection
1Department of Biomedical Sciences, Cornell University
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.
Published April 3, 2011. Keywords: Immunology, mouse, thymic lymphoma, Atm, p53, T-cell lines
JoVE Immunology and Infection
1Department of Microbiology and Immunology, The Geisel School of Medicine at Dartmouth
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.
Published July 12, 2013. Keywords: Infectious Diseases, Genetics, Microbiology, Infection, Medicine, Immunology, Molecular Biology, Cellular Biology, Biomedical Engineering, Bioengineering, Genomics, Parasitology, Pathology, Apicomplexa, Coccidia, Toxoplasma, Genetic Techniques, Gene Targeting, Eukaryota, Toxoplasma gondii, genetic manipulation, gene targeting, gene deletion, gene replacement, gene tagging, homologous recombination, DNA, sequencing
JoVE Immunology and Infection
1Department of Biological Sciences, Hunter College and Graduate Center, City University of New York, 2Sunnybrook Research Institute, Department of Immunology, University of Toronto
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.
Published October 14, 2014. Keywords: Immunology, mouse, embryonic stem cells, in vitro differentiation, OP9 cells, Delta-like 1 (Dll-1) ligand, Notch, hematopoiesis, lymphocytes, T cells
1Department of Molecular & Integrative Physiology, University of Michigan, 2Department of Internal Medicine, Division of Gastroenterology, University of Michigan
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.
Published May 17, 2013. Keywords: Cancer Biology, Medicine, Molecular Biology, Cellular Biology, Biomedical Engineering, Anatomy, Physiology, Genetics, Oncology, Surgery, Organoids, Tumor Cells, Cultured Colonic Neoplasms, Primary Cell Culture, Colon tumor, chelation, collagenase, matrigel, organoid, EGF, colon cancer, cancer, tumor, cell, isolation, immunohistochemistry, mouse, animal model
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
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.
Published November 14, 2013. Keywords: Neuroscience, Neural Stem Cells (NSCs), Body Weight, Radiotherapy, Image-Guided, Metabolism, Energy Metabolism, Neurogenesis, Cell Proliferation, Neurosciences, Irradiation, Radiological treatment, Computer-tomography (CT) imaging, Hypothalamus, Hypothalamic Proliferative Zone (HPZ), Median Eminence (ME), Small Animal Radiation Research Platform (SARRP)
1Department of Biochemistry, University of California - Riverside
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.
Published July 22, 2008. Keywords: Cellular Biology, chromatin purification, chromatin immunoprecipitation, magnetic beads, vertebrates
1Klinik und Poliklinik für Anästhesiologie, University of Wurzburg
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.
Published August 29, 2012. Keywords: Immunology, Neuroscience, Blood-brain barrier, in vitro cell culture models, brain, microvascular endothelial cells, immortalization, cEND
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
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.
Published December 4, 2014. Keywords: Neuroscience, bipolar disorder, lithium therapy, nasal biopsy, olfactory epithelium, laser-capture microdissection, real-time PCR, GSK-3β
1Department of Biomedical Genetics, Center for Oral Biology, James P Wilmot Cancer Center, University of Rochester
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.
Published June 8, 2010. Keywords: Cellular Biology, Trophoblast stem cell, trophectoderm, trophoblast giant cell, blastocyst, extraembryonic development
1Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology
This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.
Published April 7, 2008. Keywords: Cell Biology, Reprogramming, inducible lentiviruses, iPS cells, MEFs, ES cells, virus transduction, doxycycline
1Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, 2IRCCS Fondazione Santa Lucia
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.
Published June 21, 2014. Keywords: Bioengineering, Tissues, Cells, Embryonic Structures, Musculoskeletal System, Musculoskeletal Diseases, hESC, epinegetics, Skeletal Myogenesis, Myosphere, Chromatin, Lentivirus, Infection
JoVE Immunology and Infection
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
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.
Published December 18, 2012. Keywords: Cancer Biology, Stem Cell Biology, Immunology, Biomedical Engineering, Medicine, Bioengineering, Genetics, Oncology, Humanized mice, stem cell transplantation, stem cells, in vivo animal imaging, T cells, cancer, animal model
1Department of Anatomy and Cell Biology, Wayne State University School of Medicine, 2Department of Opthalmology, Wayne State University School of Medicine
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.
Published October 24, 2013. Keywords: Neuroscience, Zebrafish, Retinal Degeneration, Retina, Photoreceptor, Müller glia, Light damage
1Humanitas Clinical and Research Center, Italy, 2Institute of Genetic and Biomedical Research (IRGB), National Research Council (CNR)
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.
Published June 28, 2013. Keywords: Stem Cell Biology, Developmental Biology, Molecular Biology, Cellular Biology, Medicine, Bioengineering, Biomedical Engineering, Genetics, Cardiology, Stem Cell Research, Cardiovascular Diseases, Human cardiomyocytes, iPS cells, induced pluripotent stem cells, stem cells, cardiac differentiation, disease modeling, embryoid bodies, cell lines, cell culture
JoVE Immunology and Infection
1Department of Pharmaceutical Sciences, University of Michigan, 2Biointerfaces Institute, University of Michigan, 3Department of Biomedical Engineering, University of Michigan
We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.
Published April 29, 2015. Keywords: Immunology, nanoparticle, vaccine, biomaterial, subunit antigen, adjuvant, cytotoxic CD8+ T lymphocyte, whole animal imaging, tetramer staining, and lymph node
Science Education: General Laboratory Techniques
JoVE Science Education
Histology is the study of cells and tissues, which is typically aided by the use of a light microscope. The preparation of histological samples can vary greatly based on the inherent properties of the samples such as size and hardness as well as expected post-processing which includes planned staining techniques or other down-stream applications. As described in this video, specimen preparation typically begins with a fixation procedure to prevent degradation of the sample by naturally occurring enzymes that are released by the cells upon death. Once fixed, samples are placed into an embedding medium that is able to sufficiently support the sample. Most commonly this is paraffin wax, but other materials such as a glycerin based freezing medium and agars are also used to surround the sample during sectioning. Sectioning then takes place on a microtome or other cutting device that allows the user to shave the sample into thin slices ranging from a few microns to a few millimeters in thickness. Once cut, sections are mounted on a glass slide and stained to bring out specific features of the sample before being imaged on a microscope.…
JoVE Application Notes
1BioLife Solutions, Inc.
CryoStor cryopreservation solutions are used to prepare and preserve cells in ultra low temperature environments, without the need for serum, proteins, or high levels of cytotoxic agents.
Published October 7, 2010. Keywords: Cell Biology, Cell Culture, Freezing Medium, Cryopreservation.
JoVE Immunology and Infection
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
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.
Published November 8, 2011. Keywords: Immunology, Epstein-Barr virus, EBV, lymphoblastoid cell lines, LCL, transformation, immortalization, PBMC
1Department of Molecular and Cell Biology, Harvard
Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.
Published October 12, 2006. Keywords: Cellular Biology, Embryonic Stem Cells, ES, Tissue Culture, Freezing