JoVE General
Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast
Department of Plant Sciences, Tel Aviv University
In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.
JoVE Immunology and Infection
Tractable Mammalian Cell Infections with Protozoan-primed Bacteria
Department of Molecular Microbiology & Immunology, Oregon Health & Science University
This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.
JoVE Immunology and Infection
Purification of Pathogen Vacuoles from Legionella-infected Phagocytes
Max von Pettenkofer-Institut, Ludwig-Maximilians-Universität
This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.
JoVE General
Chronic Salmonella Infected Mouse Model
Department of Medicine, University of Rochester
Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks.
JoVE General
Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)
1Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, 2Department of Microbiology and Immunology, McGill University, 3Department of Medicine, Division of Experimental Medicine, McGill University
A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.
JoVE Immunology and Infection
Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells
1Pathology and Laboratory Medicine, University of Wisconsin-Madison, 2Pathobiological Sciences, University of Wisconsin-Madison
Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.
JoVE Immunology and Infection
Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays
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 General
Quantifying Agonist Activity at G Protein-coupled Receptors
1Department of Pharmacology, University of California, Irvine, 2Department of Pharmacology, University of California, 3Schmid College of Science, Chapman University
A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.
JoVE Immunology and Infection
Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis
Department of Microbiology and Immunology, University of Rochester
The frog Xenopus laevis provides an attractive alternative non-mammalian model for exploring the ability of heat shock protein such as gp96 to promote antigen-specific CD8 T cell responses. We present methods to study in vivo facilitation of cross-presentation of skin and tumor antigens by gp96.
JoVE Immunology and Infection
A Visual Assay to Monitor T6SS-mediated Bacterial Competition
We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.
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
Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry
1Plant Molecular and Cellular Biology Program, University of Florida, 2Department of Biology, University of Florida, 3Interdisciplinary Center for Biotechnology Research, University of Florida, 4Genetics Institute, University of Florida
Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.
JoVE Immunology and Infection
Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles
Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.
JoVE Neuroscience
Appetitive Associative Olfactory Learning in Drosophila Larvae
1Department of Biology, University of Konstanz, 2Department of Biology, University of Fribourg
Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.
JoVE General
Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay
Medical Research Council, Clinical Sciences Centre, Imperial College, Hammersmith Hospital
Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.
JoVE Immunology and Infection
Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity
1Department of Molecular Biology, University of Salzburg, 2Department of Neurology, Paracelsus Medical University, 3Department of Dermatology, Paracelsus Medical University, 4Bühlmann Laboratories, 5Christian Doppler Laboratory for Allergy Diagnosis and Therapy, University of Salzburg
Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.
JoVE General
IP-FCM: Immunoprecipitation Detected by Flow Cytometry
Department of Immunology, College of Medicine, Mayo Clinic
The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.
JoVE Immunology and Infection
Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation
Center for Translational Systems Biology and Department of Neurology, Mount Sinai School of Medicine
We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.
JoVE Immunology and Infection
Generation of Induced Regulatory T Cells from Primary Human Naïve and Memory T Cells
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 General
Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes
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 General
Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture
Department of Physiology and Biophysics, University of California, Irvine (UCI)
Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture.
JoVE General
In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes
1Department of Biochemistry & Molecular Biology, Wayne State University School of Medicine, 2Cardiovascular Research Institute, Wayne State University School of Medicine, 3Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine
Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.
JoVE Neuroscience
Neural Explant Cultures from Xenopus laevis
Department of Cell Biology, Harvard Medical School
Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.
JoVE Immunology and Infection
Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry
Department of Chemistry, Stony Brook University
Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.
JoVE Immunology and Infection
Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge
1Department of Medicine, Section of Cardiology, St. John-Providence Health System, 2Department of Immunology and Microbiology, Wayne State University School of Medicine
Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.
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 General
Imaging Cell Shape Change in Living Drosophila Embryos
1Program in Cell & Molecular Biology, Baylor College of Medicine (BCM), 2Verna & Marrs McLean Department of Biochemistry & Molecular Biology, Baylor College of Medicine (BCM)
Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.
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.
JoVE General
Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets
Department of Molecular Microbiology, University of Texas Southwestern Medical Center
The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.
JoVE Immunology and Infection
Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells
1Department of Chemical and Biomolecular Engineering, University of Houston, 2Division of Pediatrics, Research Unit 907, University of Texas MD Anderson Cancer Center
We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.
JoVE Immunology and Infection
Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes
Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.
JoVE Immunology and Infection
Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation
Department of Surgery, University of Wisconsin-Madison, School of Medicine and Public Health
We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.
JoVE General
Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH
1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behavior, University of California, Irvine (UCI)
Here we demonstrate a method for inducing and recording the progress of a delayed type-hypersensitivity (DTH) reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the effector / memory T cell response.
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
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 Immunology and Infection
Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques
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 General
A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ
1Department of Microbiology, Medical School, University of Minnesota, 2Department of Veterinary and Biomedical Sciences, University of Minnesota
A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.
JoVE General
Analyzing Large Protein Complexes by Structural Mass Spectrometry
Department of Biological Chemistry, Weizmann Institute of Science
Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.
JoVE Immunology and Infection
Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants
1Boyce Thompson Institute for Plant Research, 2Plant Pathology and Plant-Microbe Biology, Cornell University
A cell death-based assay for PTI in Nicotiana benthamiana plants is described.
JoVE Immunology and Infection
Non-invasive Imaging of Leukocyte Homing and Migration in vivo
1Department of Pathology and Immunology, Washington University in St. Louis, 2National Institute of Neurological Disorders and Stroke, NINDS, NIH - National Institute of Health
Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.
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 Neuroscience
Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain
Department of Genetics, St Jude Children's Research Hospital
This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.
JoVE General
Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method
1Chemistry Research and Development, Luminex Corporation, 2Global Marketing, Luminex Corporation
An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.
JoVE Bioengineering
In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen
1Department of Chemistry, University of Toronto, 2Institute for Optical Sciences, University of Toronto
Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.
JoVE Neuroscience
Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)
The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.
JoVE Immunology and Infection
HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL
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 Immunology and Infection
Expansion of Human Peripheral Blood γδ T Cells using Zoledronate
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
Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells
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 General
A Method For Production of Recombinant mCD1d Protein in Insect Cells.
Department of Developmental Immunology, La Jolla Institute for Allergy and Immunology
A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.
JoVE General
Visualizing Single-molecule DNA Replication with Fluorescence Microscopy
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School
This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.
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