JoVE General
Preparation of 2-dGuo-Treated Thymus Organ Cultures
MRC Center for Immune Regulation, University of Birmingham
This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.
JoVE General
Reaggregate Thymus Cultures
MRC Center for Immune Regulation, University of Birmingham
In this video the preparation of 2-dGuo-treated reaggregate thymus cultures is demonstrated.
JoVE Immunology and Infection
Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy
Laboratory of Immune Regulation, Instituto Gulbenkian de Ciência
We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of “niches” within the thymus where T cell development occurs.
JoVE General
In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy
Department of Molecular and Cell Biology, University of California, Berkeley
We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.
JoVE Immunology and Infection
Examination of Thymic Positive and Negative Selection by Flow Cytometry
Department of Medical Microbiology and Immunology, University of Alberta
We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.
JoVE Immunology and Infection
A Method for Labeling Vasculature in Embryonic Mice
1Department of Cellular Biology, University of Georgia, 2Centre for Immunology and Infection, Department of Biology and HYMS, University of York, 3Department of Genetics, University of Georgia
This article describes a method for labeling embryonic skin and thymus blood vessels.
JoVE General
Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats
Department of Physiology and Biophysics, University of California, Irvine (UCI)
Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.
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
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 Immunology and Infection
Competitive Homing Assays to Study Gut-tropic T Cell Migration
Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School
Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.
JoVE General
Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice
1Research Animal Diagnostic Services, Charles River, 2Research Models and Services, Charles River, 3Department of Comparative Medicine, University of Washington
This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.
JoVE Immunology and Infection
Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies
1Molecular and Cell Biology Program, Ohio University, 2Department of Biomedical Sciences, College of Osteopathic Medicine, Ohio University, 3Department of Biomedical Engineering, Russ College of Engineering and Technology, Ohio University
Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.
JoVE Bioengineering
Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture
ARTORG Center for Biomedical Engineering, University of Bern
This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.
JoVE Neuroscience
Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction
1Lehrstuhl für Biomolekulare Sensoren, Technische Universität München, 2Center for Integrated Protein Science (Munich) at the Institute of Neuroscience, Technische Universität München, 3TUM Institute for Advanced Study and German Center for Neurodegenerative Diseases, Technische Universität München, 4Munich Cluster for Systems Neurology (SyNergy), Technische Universität München
Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.
JoVE Clinical and Translational Medicine
Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice
Department of Anesthesiology, University of Colorado
A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.
JoVE Clinical and Translational Medicine
Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging
1Department of Pathology, Center for Cardiovascular Biology, and Institute for Stem Cell and Regenerative Medicine, University of Washington, 2Departments of Bioengineering and Medicine/Cardiology, University of Washington
Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (μCT) imaging.
JoVE Immunology and Infection
Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice
1Department of Environmental Medicine, New York University School of Medicine, Tuxedo, 2Division of Allergy, Pulmonary, & Critical Care Medicine, Department of Medicine, Vanderbilt University Medical Center, 3Division of Pulmonary Medicine, New York University School of Medicine
A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.
JoVE Chemistry
Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples
Department of Chemistry, University of Virginia
A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.
JoVE Bioengineering
Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts
Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago
Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.
JoVE Clinical and Translational Medicine
Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy
Department of Anesthesiology & Pain Medicine, University of Washington School of Medicine
Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.
JoVE General
Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish
1Department of Molecular Pathology, Massachusetts General Hospital, Harvard Medical School, 2Department of Molecular Pathology, Massachusetts General Hospital Cancer Center, Harvard Stem Cell Institute
Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.
JoVE Neuroscience
Whole Animal Perfusion Fixation for Rodents
1Biomedical Engineering, University of Michigan, 2Department of Neurological Surgery, University of Washington School of Medicine
Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain.
JoVE General
Making Gynogenetic Diploid Zebrafish by Early Pressure
1Institute of Neuroscience, University of Oregon, 2Division of Basic Science, Fred Hutchinson Cancer Research Center - FHCRC
This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.
JoVE Clinical and Translational Medicine
An in vivo Assay to Test Blood Vessel Permeability
We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.
JoVE Bioengineering
In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage
1Neurosurgery, Baylor College of Medicine, 2Michael E. DeBakey Veterans Affairs Medical Center, 3Molecular & Cellular Biology, Baylor College of Medicine
We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.
JoVE Immunology and Infection
Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies
1Research Group Immune Regulation, Helmholtz Centre for Infection Research, 2Research Group Infection Immunology, Institute of Medical Microbiology, Otto-von-Guericke University, 3Department of Experimental Immunology, Helmholtz Centre for Infection Research
We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.
JoVE General
A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants
1Department of Experimental Oncology, European Institute of Oncology, 2Department of Pathology and Laboratory Medicine, European Institute of Oncology, 3U.O. Gastroenterologia 2, IRCCS Ca' Granda, Ospedale Policlinico di Milano
We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.
JoVE Immunology and Infection
Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection
Department of Microbiology, Immunology and Pathology, Colorado State University
Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.
JoVE Immunology and Infection
Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry
1Department of Immunology, University of Colorado School of Medicine, 2Division of Cell Biology, Department of Pediatrics, National Jewish Health, 3Department of Microbiology, Immunology, and Pathology, Colorado State University, 4Department of Immunology, National Jewish Health
We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.
JoVE Immunology and Infection
Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation
1Institute of Immunology, Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 2Pathobiology, School of Veterinary Medicine, University of Pennsylvania
Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.
JoVE General
Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer
Shimadzu, Scientific Instruments
This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.
JoVE General
Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.
1Department of Otology and Laryngology, Harvard Medical School, 2Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, 3Department of Communication Sciences and Disorders, Emerson College, 4Program in Speech and Hearing Bioscience and Technology, Division of Health Science and Technology, Harvard
This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.
JoVE Immunology and Infection
Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes
1Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), 2Inserm, U1016, Paris, France
This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.
JoVE Immunology and Infection
Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells
This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.
JoVE Neuroscience
Strategies for Study of Neuroprotection from Cold-preconditioning
Department of Neurology, The University of Chicago Medical Center
We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.
JoVE Immunology and Infection
Retroviral Transduction of T-cell Receptors in Mouse T-cells
1NYU Cancer institute, New York University School of Medicine, 2Program in Structural Biology, New York University School of Medicine
We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction
JoVE Immunology and Infection
A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells
1Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Tsukuba, Japan, 2Safety Research Team, National Institute of Animal Health, Tsukuba, Japan
A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.
JoVE General
Dissection of the Adult Zebrafish Kidney
Department of Biological Sciences, University of Notre Dame
The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.
JoVE Clinical and Translational Medicine
Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis
1University Heart Center Hamburg, TSI-Lab, Germany, 2Cardiovascular Research Center, University of Hamburg, 3Department of Medicine, Cardiology Division, Pulmonary Hypertension Program, University of Alberta, 4Department of Medicine, Stanford University School of Medicine, 5Department of Biomedical Sciences, Institute of Physiology, Pathophysiology, and Biophysics, University of Veterinary Medicine, Vienna, 6Translumina GmbH, Hechingen, 7Department of Cardiothoracic Surgery, Stanford University School of Medicine
This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.
JoVE General
Transverse Aortic Constriction in Mice
1Department of Molecular Physiology and Biophysics, Baylor College of Medicine (BCM), 2The Margaret M. and Albert B. Alkek Department of Medicine, Baylor College of Medicine (BCM)
Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.
JoVE General
Preparing T Cell Growth Factor from Rat Splenocytes
Department of Physiology and Biophysics, University of California, Irvine (UCI)
We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.
JoVE Neuroscience
Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection
1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2Department of Microbiology & Immunology, Medical University of South Carolina, 3National Institute on Deafness and Other Communication Disorders, National Institutes of Health
Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.
JoVE Immunology and Infection
Mass Spectrometric Analysis of Glycosphingolipid Antigens
1Undergraduate Program, Rice University, 2Proteomics Facility, Department of Pathology, University of Texas MD Anderson Cancer Center, 3Department of Melanoma Medical Oncology, University of Texas MD Anderson Cancer Center, 4University of Texas Graduate School of Biological Sciences at Houston
A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.
JoVE General
Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney
1Department of Molecular Medicine, Maine Medical Center Research Institute, 2Molecular Medicine and Gene Therapy, Lund University Hospital
In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.
JoVE Immunology and Infection
Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples
Department of Basic Sciences, Mississippi State University
Here we describe a procedure allowing the labeling of Edwardsiella ictaluri in situ in histological sections from channel catfish Ictalurus punctatus using indirect immunohistochemistry with monoclonal antibodies Ed9 as a primary, and fluorescent FitC labeled antibodies as a secondary. This allowed for the detection of the bacterium using fluorescent microscopy.
JoVE Neuroscience
Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti
Department of Pediatrics, Children’s Research Institute, Medical College of Wisconsin
A simple procedure of vibratome sectioning the organ of Corti, followed by immunohistochemistry and confocal microscopy is described. This procedure allows for improved preservation of the fine cytoarchitecture of the mammalian organ of Corti, and consequently allows for accurate quantification of cell types.
JoVE Bioengineering
Photoacoustic Cystography
1Department of Biomedical Engineering, University at Buffalo, The State University of New York, 2Department of Creative IT Engineering, Pohang University of Science and Technology (POSTECH), 3School of Electrical Engineering and Computer Science, Kyungpook National University
Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.
JoVE Neuroscience
Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine
In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.
JoVE General
Fruit Volatile Analysis Using an Electronic Nose
1Department of Plant Sciences, University of California, Davis, 2Department of Chemical Engineering and Material Science, University of California, Davis, 3Department of Viticulture and Enology, University of California, Davis
A rapid method for volatile compound analysis in fruit is described. The volatile compounds present in the headspace of a homogenate of the sample are rapidly separated and detected with ultra-fast gas chromatography (GC) coupled with a surface acoustic wave (SAW) sensor. A procedure for data handling and analysis is also discussed.
JoVE General
Dissection of Organs from the Adult Zebrafish
Department of Cell and Developmental Biology, University of Pennsylvania-School of Medicine
This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.
JoVE Bioengineering
Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves
Department of Agricultural and Biological Engineering, Mississippi State University
A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.
More Results...
