The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.

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 JoVE General

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers


JoVE 1167 3/06/2009

1Tetramer Core Laboratory, Benaroya Research Institute, 2Kwok Laboratory, Benaroya Research Institute, 3Nepom Laboratory, Benaroya Research Institute

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

 JoVE Immunology and Infection

Isolation of Functional Cardiac Immune Cells


JoVE 3020 12/05/2011

Department of Cell Biology and Anatomy, University of South Carolina- School of Medicine

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

 JoVE Clinical and Translational Medicine

The α-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva


JoVE 3999 5/16/2012

1Department of Medicine, Weill Cornell Medical College, 2Department of Oral Biology, University of Missouri-Kansas City-School of Dentistry, 3Department of Pharmacology and Toxicology, University of Missouri Kansas City- School of Pharmacy, 4Regional Hospital, Bamenda, NWP, Cameroon, 5Mezam Polyclinic HIV/AIDS Treatment Center, Cameroon, 6Institute for Human Genetics and Biochemistry

A CD4 enumeration method, the α-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The α-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

 JoVE Immunology and Infection

Generation of Induced Regulatory T Cells from Primary Human Naïve and Memory T Cells


JoVE 3738 4/16/2012

Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky

We describe a method for generating regulatory, memory and naïve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

 JoVE Immunology and Infection

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy


JoVE 2061 10/07/2010

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 Immunology and Infection

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays


JoVE 2668 3/14/2011

Department of Immunology and Microbiology, Rush University Medical Center

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 JoVE Immunology and Infection

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques


JoVE 1743 4/22/2010

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

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

 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


JoVE 2381 1/14/2011

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

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes


JoVE 3986 5/14/2012

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

Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model


JoVE 2312 10/31/2010

1National Health Laboratory Services (NHLS-SA), 2Department of Molecular Medicine and Haematology, University of Witwatersrand, 3Lightcurve Films

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

 JoVE Immunology and Infection

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance


JoVE 3071 7/22/2011

1Department of Pediatrics/Allergy, Medical College of Wisconsin, 2Flow Cytometry Core Facility, Medical College of Wisconsin, 3Max McGee National Research Center for Juvenile Diabetes and Human Molecular Genetics Center, Medical College of Wisconsin

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

 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


JoVE 50070 2/01/2013

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

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes


JoVE 4287 12/13/2012

1Department of Flow and Image Cytometry, Roswell Park Cancer Institute, 2Flow Cytometry & Cell Sorting Resource Laboratory, University of Pennsylvania, 3SciGro, Inc., 4Department of Pathology and Laboratory Medicine, University of Pennsylvania

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

 JoVE Immunology and Infection

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant


JoVE 2736 5/27/2011

Center for Cell and Gene Therapy, Baylor College of Medicine

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

 JoVE Immunology and Infection

Isolation of Lymphocytes from Mouse Genital Tract Mucosa


JoVE 4391 9/03/2012

1Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, 2California NanoSystems

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

 JoVE Immunology and Infection

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells


JoVE 4420 10/22/2012

Center for Immunology and Inflammatory Diseases, and Pulmonary and Critical Care Unit, Massachusetts General Hospital and Harvard Medical School

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 General

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells


JoVE 3159 10/26/2011

1Charles C. Gates Regenerative Medicine and Stem Cell Biology Program, University of Colorado Denver, 2Department of Medicine, University of Colorado Denver, 3Cancer Center, University of Colorado Denver, 4Webb Waring Institute, University of Colorado Denver

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

 JoVE Immunology and Infection

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus


JoVE 3627 5/07/2012

1Center for Cell and Gene Therapy, Baylor College of Medicine, 2Pathology and Immunology, Baylor College of Medicine, 3Department of Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, 4Medicine, Baylor College of Medicine, 5Department of Pediatrics, Baylor College of Medicine

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly naîve T cells.

 JoVE General

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors


JoVE 3736 7/08/2012

1Institute for Research in Biomedicine, Bellinzona (Switzerland), 2Dipartimento di Biologia e Genetica per le Scienze Mediche, Universitá degli Studi di Milano

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

 JoVE Immunology and Infection

piggyBac Transposon System Modification of Primary Human T Cells


JoVE 4235 11/05/2012

1Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, 2Department of Medicine, Division of Nephrology, Baylor College of Medicine, 3Department of Immunology and Pathology, Shinshu University School of Medicine, 4Center for Cell and Gene Therapy, Baylor College of Medicine, 5Department of Pediatrics, Baylor College of Medicine, 6Program in Cell and Molecular Biology, Baylor College of Medicine, 7Department of Molecular Virology and Microbiology, Baylor College of Medicine, 8Michael E. DeBakey VA Medical Center

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

 JoVE Immunology and Infection

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers


JoVE 3757 3/08/2012

1Department of Pathology, New York University Langone School of Medicine, 2Program in Molecular Pathogenesis, Marty and Helen Kimmel Center for Biology and Medicine and Skirball Institute for Biomolecular Medicine, 3Laboratory of Molecular Immunogenetics, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 4Veteran Affairs New York Harbor Healthcare System

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

 JoVE Immunology and Infection

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood


JoVE 3741 4/16/2012

Department of Internal Medicine, Yale University School of Medicine

We describe use of ImageStream technology (www.amnis.com), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

 JoVE Immunology and Infection

Expansion of Human Peripheral Blood γδ T Cells using Zoledronate


JoVE 3182 9/09/2011

1Department of Immunotherapeutics (Medinet), University of Tokyo Hospital, 2MEDINET Co., Ltd

A method to expand γδ T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived γδ T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of γδ T cells can be applied to autologous cellular immunotherapy of cancer.

 JoVE General

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces


JoVE 1969 3/19/2010

Institute of Biophysics, Johannes Kepler Universitat Linz

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

 JoVE Immunology and Infection

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT


JoVE 2168 9/30/2010

1 , Whitehead Institute for Biomedical Research, 2Departments of Microbiology and Biological Sciences, National University of Singapore, 3Department of Biology, Massachusetts Institute of Technology

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

 JoVE Immunology and Infection

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein


JoVE 2198 9/23/2010

National Center for Biodefense and Infectious Diseases, Department of Molecular and Microbiology, George Mason University

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

 JoVE General

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging


JoVE 773 7/09/2008

1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behaviour, University of California, Irvine (UCI)

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

 JoVE Immunology and Infection

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses


JoVE 2146 11/28/2010

1Penn Ovarian Cancer Research Center, Center for Research on Reproduction and Womans Health, Department of Obstetrics and Gynecology, University of Pennsylvania-School of Medicine, 2Women's Cancer Program, Fox Chase Cancer Center

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

 JoVE Immunology and Infection

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis


JoVE 2595 2/24/2011

1Department of Kinesiology, Recreation, and Sport, Western Kentucky University, 2Department of Health and Human Performance, University of Houston

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

 JoVE Immunology and Infection

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice


JoVE 50112 1/02/2013

The Walter and Eliza Hall Institute of Medical Research

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

 JoVE Immunology and Infection

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL


JoVE 2801 4/11/2011

1Immunology Graduate Program, Johns Hopkins University, 2Department of Internal Medicine, Far-Eastern Memorial Hospital, 3Department of Pathology, Johns Hopkins University, 4Institute of Cell Engineering, Johns Hopkins University

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

 JoVE Clinical and Translational Medicine

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice


JoVE 3644 2/03/2012

Pharmazentrum Frankfurt / ZAFES, Goethe University Hospital Frankfurt

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

 JoVE Immunology and Infection

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


JoVE 4181 12/18/2012

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

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging


JoVE 3123 7/28/2011

1Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, 2Department of Mechanical and Aerospace Engineering, The Ohio State University, 3Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, 4Dept. of Chemical and Biomolecular Engineering, Vanderbilt University

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

 JoVE General

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass


JoVE 50064 5/01/2013

Department of Pathology, University of Utah School of Medicine

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

 JoVE Clinical and Translational Medicine

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma


JoVE 50200 3/13/2013

1Institute of Pathology, Laboratory of Molecular Tumor Pathology, Charité - Universitätsmedizin Berlin, 2Institute for Chemistry and Biochemistry, Free University Berlin, 3Laboratory for Functional Genomics Charité (LFGC), Charité - Universitätsmedizin Berlin, 4Comprehensive Cancer Center Charité, Charité - Universitätsmedizin Berlin

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

 JoVE Immunology and Infection

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine


JoVE 4040 5/21/2012

1Department of Pediatrics, Emory University, 2Department of Pathology & Laboratory Medicine, Emory University

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

 JoVE Immunology and Infection

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood


JoVE 50402 4/16/2013

1Department of Pathology and Anatomical Sciences, University of Missouri-Columbia, 2Laboratory for Infectious Disease Research, University of Missouri-Columbia

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

 JoVE Immunology and Infection

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells


JoVE 4333 12/29/2012

Department of Microbiology and Immunology, University of Maryland

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

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