JoVE Immunology and Infection
Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy
1School of Chemistry, Food and Pharmacy, The University of Reading, 2Department of Nutritional Sciences, The University of Reading
A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.
JoVE Neuroscience
Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction
1San Diego Regenerative Medicine Institute, 2Xcelthera, 3Department of Neurosurgery, Harvard Medical School, 4Division of SCI Research, VA Boston Healthcare System, 5Program in Stem Cell & Regenerative Biology, Sanford-Burnham Medical Research Institute, 6La Jolla IVF
We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.
JoVE General
Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines
Department of Surgery, Weill Cornell Medical College
A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.
JoVE Neuroscience
Revealing Neural Circuit Topography in Multi-Color
We provide a practical guide for delivering tracers in vivo and use the spinocerebellar pathway as a model system to demonstrate essential steps for successful neuronal circuit analysis in mice. We describe in detail our versatile tracing protocol that exploits wheat germ agglutinin (WGA) conjugated to Alexa fluorophores.
JoVE General
RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus
Biology Department, California State University
In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.
JoVE Neuroscience
A Molecular Readout of Long-term Olfactory Adaptation in C. elegans
1Department of Biological Sciences and Institute for Neuroscience, George Washington University, 2Fred Hutchinson Cancer Research Center, 3Department of Cell and Tissue Biology, University of California San Francisco
Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.
JoVE Neuroscience
Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement
Department of Neural and Pain Sciences, University of Maryland
A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.
JoVE General
Germ Cell Transplantation and Testis Tissue Xenografting in Mice
Department of Comparative Biology and Experimental Medicine, University of Calgary
Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.
JoVE Clinical and Translational Medicine
Teratoma Generation in the Testis Capsule
1Department of Chemical Physiology, Scripps Research Institute, 2Department of Chemical Physiology, Scripps Research Institute, 3Department of Reproductive Medicine, University of California
Human pluripotent stem cells (hPSCs) have the potential to treat a myriad of different diseases. The utility of these cells lies in the fact that they can differentiate into any cell type in the body. Here we describe the teratoma assay, which is used to demonstrate the pluripotence of hPSCs.
JoVE General
Generating Chimeric Zebrafish Embryos by Transplantation
HHMI and Division of Basic Sciences, Fred Hutchinson Cancer Research Center - FHCRC
A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.
JoVE General
Upright Imaging of Drosophila Embryos
1Department of Biology, Case Western Reserve University, 2Department of Genetics, Case Western Reserve University
Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.
JoVE General
Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution
1Department of Cellular and Molecular Medicine, University of California, San Diego, 2Biosecurity and Public Health, Los Alamos National Laboratory
Unfixed frozen tissue samples embedded in Optimal Cutting Temperature medium (OCT) can be used to study natural distribution and glycosylation of secreted mucus. In this approach tissue processing is minimal and the natural presentation of glycolipids, mucins and glycan-epitopes is preserved. Tissue sections can be analyzed by immunohistochemistry using fluorescence or chromogenic detection.
JoVE General
Basic Caenorhabditis elegans Methods: Synchronization and Observation
1Department of Cancer and Human Molecular Genetics, Bellvitge Institute for Biomedical Research, 2C. elegans Core Facility, Bellvitge Institute for Biomedical Research
The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.
JoVE General
Chromatin Immunoprecipitation (ChIP) using Drosophila tissue
Department of Biology, Johns Hopkins University
Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.
JoVE General
Isolation and Derivation of Mouse Embryonic Germinal Cells
The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.
JoVE General
Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation
Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.
JoVE General
Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay
Department of Developmental and Cell Biology, University of California, Irvine (UCI)
This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.
JoVE Bioengineering
A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids
Department of Surgery, UMDNJ-Robert Wood Johnson Medical School
We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.
JoVE General
Fertilization of Xenopus oocytes using the Host Transfer Method
Department of Biology, University of Iowa
Procedure for fertilizing Xenopus oocytes by the host transfer method.
JoVE General
Dissection and Staining of Drosophila Larval Ovaries
Department of Biological Regulation, Weizmann Institute of Science
How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.
JoVE Chemistry
Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development
ISOF - Bio Free Radicals, Consiglio Nazionale delle Ricerche
Radical-based biomimetic chemistry has been applied to building-up libraries necessary for biomarker development.
JoVE General
Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.
Department of Genetics, Yale University School of Medicine
Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.
JoVE Immunology and Infection
Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells
Laboratory for Epithelial Immunobiology, RIKEN Research Center for Allergy and Immunology
M cells in a specialized follicle-associated epithelium covering Peyer’s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.
JoVE General
In-vivo Centrifugation of Drosophila Embryos
Department of Biology, University of Rochester
We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.
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 General
Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::β-glucuronidase Reporter Gene Expression
Department of Cell Biology and Molecular Genetics, University of Maryland College Park
This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::β-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.
JoVE Immunology and Infection
Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence
Department of Bacteriology, University of Wisconsin-Madison
The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.
JoVE General
An Introduction to Worm Lab: from Culturing Worms to Mutagenesis
Department of Biology, University of Texas at Arlington
Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.
JoVE Clinical and Translational 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
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 Clinical and Translational Medicine
Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs
1Department of Neurosurgery, Emory University, 2Department of Neuroscience, Medical University of South Carolina, 3Division of Neurosurgery, University of Alabama, Birmingham, 4Department of Biomedical Engineering, Georgia Institute of Technology, 5Department of Biomedical Engineering, Emory University
Short visual description of the surgical technique and device used for the delivery of (gene and cell) therapies into the spinal cord. The technique is demonstrated in the animal but is entirely translatable and currently being used for human application.
JoVE General
Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI
We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.
JoVE General
Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1
1Department of Biomedical Informatics, The Ohio State University, 2Departments of Molecular Immunology and Clinical Oncology, Tohoku University
We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.
JoVE General
The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol
Department of Biology, University of Waterloo
A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.
JoVE General
Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization
Department of Molecular, Cellular and Developmental Biology, Yale University
Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.
JoVE Bioengineering
Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and and Induced Pluripotent Stem Cells (iPSCs)
1Department of Medicine (Hematology, Oncology, and Transplant), University of Minnesota, Minneapolis, 2Stem Cell Institute, University of Minnesota, Minneapolis
This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.
JoVE General
ES Cell-derived Neuroepithelial Cell Cultures
McLean Hospital, Harvard Medical School
Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).
JoVE General
Flow Cytometry Purification of Mouse Meiotic Cells
1Genome Plasticity Laboratory, Department of Cancer Biology, The Scripps Research Institute, 2Flow Cytometry Core, The Scripps Research Institute
An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.
JoVE General
High-Throughput Measurement and Classification of Organic P in Environmental Samples
School of Engineering, University of Vermont
This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.
JoVE General
Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction
1San Diego Regenerative Medicine Institute, 2Xcelthera, 3Department of Neurosurgery, Harvard Medical School, 4Division of SCI Research, VA Boston Healthcare System, 5Program in Stem Cell & Regenerative Biology, Sanford-Burnham Medical Research Institute, 6La Jolla IVF
We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.
JoVE Bioengineering
Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control
1Entomology, International Center for Tropical Agriculture (CIAT), Cali, Colombia, 2Sustainable Perennial Crops Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland, USA
This protocol demonstrates two inoculation methods to introduce the fungal entomopathogen Beauveria bassiana as an endophyte in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control.
JoVE General
Operant Learning of Drosophila at the Torque Meter
Department of Neurobiology, Free University of Berlin
Measuring the yaw torque of tethered Drosophila with the torque meter allows the neuroscientist exquisite control of the stimulus situation of the experimental animal. Together with the unique genetic tools available in the fruit fly, this paradigm is used for a wide variety of neurobiological research.
JoVE General
Generation of Stable Transgenic C. elegans Using Microinjection
Department of Biological Sciences, University of Alabama
This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.
JoVE Neuroscience
A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats
Psychology, University of Toronto Scarborough
Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.
JoVE General
Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development
1Department of Biological Sciences, The George Washington University, 2Department of Anatomy and Regenerative Biology, The George Washington University
The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.
JoVE Neuroscience
A Caenorhabditis elegans Model System for Amylopathy Study
We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human Aβ42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.
JoVE General
Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia
1Division of Cardiovascular Medicine, Stanford University, 2Department of Radiology, Stanford University
The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.
JoVE Neuroscience
Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents
1Center for Molecular and Behavioral Neuroscience, University of New Jersey, 2Center for Interdisciplinary Research in Biology, Collège de France, 3Janelia Farm Research Campus, Howards Hughes Medical Institute, 4Deptartment of Psychology, University of Wisconsin at Milwaukee
We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.
JoVE Clinical and Translational Medicine
Orthotopic Small Bowel Transplantation in Rats
1Department of Surgery, University of Bonn, Germany, 2Department of Surgery, Kyoto University Hospital
Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.
JoVE Neuroscience
Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice
Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, University of Toledo
The experimental designs proposed here focus on studying the effects of alcohol exposure in apoptosis and the application of neurotrophic peptide during pregnancy in fetal brain. A detailed description from the breeding to the collection of fetal brains is described. Techniques for determination of apoptosis are also described in detail.
JoVE General
Blood Collection from the American Horseshoe Crab, Limulus Polyphemus
1Department of Molecular and Cell Biology, University of California, Davis, 2Marine Biological Laboratory - MBL- woods hole, 3Department of Biological Sciences, Hunter College of CUNY
The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and α2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.
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