Purkinje Cells:
The output neurons of the cerebellar cortex.
JoVE Neuroscience
The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures
Anatomical Institute, Department of Biomedicine, University of Basel
We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.
JoVE Neuroscience
Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection
1Laboratory of Genetics, The Salk Institute for Biological Studies, 2Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies
This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.
JoVE General
Generation of Single-Cell Suspensions from Mouse Neural Tissue
Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.
JoVE Clinical and Translational Medicine
Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model
Institute of Human Physiology and Clinical Experimental Research, Semmelweis University
We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.
JoVE Neuroscience
Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination
1Neuroscience Graduate Program, Keck School of Medicine, University of Southern California, 2Zilkha Neurogenetic Institute, University of Southern California, 3Department of Cell and Neurobiology, Neuroscience Graduate Program, Keck School of Medicine, University of Southern California
An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.
JoVE General
Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons
1Department of Genetics and Development, Columbia University, 2Department of Pathology and Cell Biology, Columbia University, 3Department of Neuroscience, Columbia University, 4Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School
Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.
JoVE Neuroscience
Wholemount Immunohistochemistry for Revealing Complex Brain Topography
1Department of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, 2Department of Cell Biology and Anatomy and the Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary
Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.
JoVE Neuroscience
Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells
Department of Cell and Developmental Biology, Vanderbilt University
This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.
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
Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)
1Institute for Clinical Neurobiology, University of Wuerzburg, 2Department of Synapses - Circuits - Plasticity, Max Planck Institute of Neurobiology, Martinsried, 3Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians University of Munich
Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.
JoVE General
Selective Capture of 5-hydroxymethylcytosine from Genomic DNA
1Department of Human Genetics, Emory University School of Medicine, 2Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago
Described is a two-step labeling process using β-glucosyltransferase (β-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.
JoVE Immunology and Infection
Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes
Department of Internal Medicine B, University Medicine Greifswald
The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.
JoVE General
Gene Transfer into Older Chicken Embryos by ex ovo Electroporation
1Albrecht-Kossel-Institute for Neuroregeneration, School of Medicine University of Rostock, 2Institute of Anatomy I, School of Medicine University of Jena
A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.
JoVE Neuroscience
Video-oculography in Mice
1Department of Neuroscience, Erasmus MC, Rotterdam, The Netherlands, 2Department of Neuroscience, Royal Dutch Academy of Arts & Sciences (KNAW)
Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.
JoVE Immunology and Infection
Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes
Department of Microbiology and Immunology, Pennsylvania State University College of Medicine
Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.
JoVE Immunology and Infection
Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors
1Division of Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research, 2Aaron Diamond AIDS Research Center, Laboratory of Retrovirology, The Rockefeller University, NY
We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.
JoVE General
Generation of Human CD40-activated B cells
In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.
JoVE Immunology and Infection
Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells
Division of Pediatrics, MD Anderson Cancer Center - University of Texas
Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.
JoVE General
Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny
1Department of Medicine, Hematology-Oncology, University of California, Los Angeles, 2Department of Biological Chemistry, University of California, Los Angeles
mStrawberry OP9 cells allow for complete evaluation of all ES-derived progeny from co-culture.
JoVE General
Isolation and Culture of Adult Epithelial Stem Cells from Human Skin
Department of Cancer Biology, University of Massachusetts Medical School
A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.
JoVE General
Measuring Replicative Life Span in the Budding Yeast
1Department of Biochemistry, University of Washington, 2Department of Pathology, University of Washington
In this article we present a general protocol for measuring the replicative life span of yeast mother cells.
JoVE General
The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF
1Research and Development, Stemgent, 2Product Marketing, Stemgent
SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.
JoVE General
Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency
Gene Expression Division, Bio-Rad Laboratories, Inc.
This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.
JoVE General
Isolation of Stem Cells from Human Pancreatic Cancer Xenografts
Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine
Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.
JoVE General
Determining Cell Number During Cell Culture using the Scepter Cell Counter
Bioscience Division, Millipore Inc
The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.
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 General
Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells
1The Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa
An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.
JoVE General
Analysis of Cell Cycle Position in Mammalian Cells
1Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, 2London Regional Cancer Program, Children's Health Research Institute, and Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario
Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.
JoVE Immunology and Infection
Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System
Department of Immunology, College of Medicine, Mayo Clinic
The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.
JoVE Neuroscience
The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry
1Department of Neurosurgery, The University of Florida, 2Laboratory for Stem Cell Research, Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.
JoVE Clinical and Translational Medicine
Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)
1Department of Neurosurgery, The University of Florida, 2Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.
JoVE Immunology and Infection
Murine Model of CD40-activation of B cells
In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.
JoVE Clinical and Translational Medicine
A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells
Lombardi Comprehensive Cancer Center, Georgetown University
This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.
JoVE General
Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs
Department of Chemical Engineering and Materials Science, City of Hope Cancer Center
Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.
JoVE Immunology and Infection
Induction and Assessment of Class Switch Recombination in Purified Murine B Cells
Department of Immunology, University of Toronto
Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.
JoVE Bioengineering
Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells
1CYTOO Cell Architects, Grenoble, France, 2Centre Commun de Quantimétrie, Faculté de Médecine Rockefeller, Lyon, France
Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.
JoVE Clinical and Translational Medicine
Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle
1Department of Radiology, Molecular Imaging Program at Stanford (MIPS), 2Stanford School of Medicine, Stanford University
We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.
JoVE General
Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences
We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.
JoVE Immunology and Infection
Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood
1Division of Pediatrics, U.T. MD Anderson Cancer Center, 2Department of Stem Cell Transplantation and Cellular Therapy, U.T. MD Anderson Cancer Center
T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.
JoVE General
Derivation of Human Embryonic Stem Cells by Immunosurgery
Department of Molecular and Cell Biology, Harvard
The ability of human embryonic stem cells to self-renew and differentiate into all cell types of the body suggests that they hold great promise for both medical applications and as a research tool for addressing fundamental questions in development and disease. Here, we provide a concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos by immunosurgical isolation of the inner cell mass.
JoVE General
Detection and Isolation of Viable Mouse IL-17-Secreting T Cells
This procedure describes the detection and isolation of mouse TH17 leukocytes that actively secrete IL-17 upon stimulation.
JoVE Clinical and Translational Medicine
Experimental Metastasis Assay
1Department of Biomedical Genetics, University of Rochester Medical Center, 2Department of Dermatology, University of Rochester Medical Center
This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.
JoVE Immunology and Infection
Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy
1Division of Infectious Diseases, Department of Medicine, Immunology Institute, Mount Sinai School of Medicine, 2NSF Center for Biophotonics, University of California, Davis, 3Structural and Computational Biology Unit, European Molecular Biology Laboratory
This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.
JoVE General
Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts
Department of Molecular and Cellular Biology, University of Guelph
In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.
JoVE Neuroscience
Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells
1Alaska Basic Neuroscience Program, Institute of Arctic Biology, University of Alaska at Fairbanks, 2Department Biochemistry, Hood College, 3Department of Cell Biology, Neuronascent, Inc., 4Research and Development, Neuronascent, Inc.
Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.
JoVE Immunology and Infection
Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation
1Department of Microbiology & Immunology, Virginia Commonwealth University- Massey Cancer Center, 2Department of Internal Medicine, Virginia Commonwealth University- Massey Cancer Center, 3Department of Surgery, Virginia Commonwealth University- Massey Cancer Center
An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.
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 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
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 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
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
Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes
Department of Immunology, Duke University
We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.
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