Springer protocols feed by Immunology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Monitoring the Intracellular Routing of Internalized Antigens by Immunofluorescence Microscopy
Professional antigen-presenting cells such as dendritic cells (DCs) and macrophages internalize extracellular antigens, process them intracellularly, and present the resulting antigen-derived peptides in the context of MHC I or MHC II molecules. Since the intracellular routing of the antigen determines whether antigens are presented on MHC I or MHC II molecules, a profound analysis of the intracellular distribution of the internalized antigens is of high interest. Here, we describe an immunofluorescence protocol to monitor the intracellular routing of the model-antigen Ovalbumin in bone marrow-derived dendritic cells (BM-D...
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Monitoring Dendritic Cell Activation and Maturation
Since the 1997 discovery that the first identified human homologue of Drosophila Toll could activate the innate immune system, the innate arm of immunity has rapidly taken on a new light as an important player in the recognition of pathogens and damaged self. The recognition of danger by dendritic cells (DC) is a crucial step in activating the adaptive immune system. Different DC express varied subsets of pattern recognition receptors (PRR), enabling both overlap and exclusivity in the recognition of danger signals by DC. PRR-mediated DC maturation and activation can be measured by changes in the surface expression of cost...
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Preparation of Dendritic Cells by In Vitro Cultures
In vitro cultures of bone marrow-derived precursors are a convenient method for generating dendritic cells (DC). This method additionally overcomes the problem of low availability of certain DC types, DC heterogeneity, and laborious procedures encountered using ex vivo isolation protocols. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
The Purification of Large Numbers of Antigen Presenting Dendritic Cells from Mouse Spleen
Dendritic cells (DC) are found at low frequency in lymphoid and non-lymphoid tissues. Different DC subsets are adept at different roles in immunity in diverse scenarios of attack by infectious agents, as well as in the maintenance of self-tolerance. A key element in the ability of DC to initiate adaptive immune responses is their capacity to capture and process antigen, whether from pathogens, vaccines or self-components, and present it to T cells. Our typical procedure for isolation of the different DC types from murine spleen involves their digestion from the tissue using collagenase, selection of cells of light density,...
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Tracking Antigen-Specific CD8+ T Cells Using MHC Class I Multimers
The tracking of epitope-specific T cells is a useful approach for the study of adaptive immune responses. This protocol describes how Major Histocompatibility Complex Class I (MHC-I) multimers can be used to stain, enrich, and enumerate (rare) populations of CD8+ T cells specific for a given antigen. It provides the detailed steps for multimer labeling, magnetic enrichment, and cytometric analysis. Additionally, it provides informations for multiplexing experiments in order to achieve simultaneous detection of multiple antigenic specificities, and strategies for coupling the protocol with functional assays (e.g., intracell...
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Production of CD4+ and CD8+ T Cell Hybridomas
T cell hybridomas are very useful tools to investigate antigen presenting cell (APC) function. They were developed based on the fusion technology that led to monoclonal antibody section. Antigen-specific primary T cells are generated and fused to an immortal thymoma line. Unfused thymoma cells are eliminated by engineered metabolic selection. Antigen-specific hybridomas are identified and may be characterized in detail. Primary T cells are preferable for studies of the regulatory mechanisms intrinsic to T cells, but for study of antigen presentation T cell hybridomas have advantages over primary T cell clones, including th...
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Cloning CD8+ Cytolytic T Lymphocytes
CD8+ T lymphocyte cloning has resulted in many fundamental advances: structural elucidation of peptide-MHC recognition, spatiotemporal dissection of the thymic positive and negative selection processes and is further expected, TCRs being the key molecules controlling T cell activation, to provide us with molecular tools of immuno-therapeutical interest for infectious, tumor, and autoimmune diseases. However, cloning CD8+ T lymphocytes remains a relatively difficult enterprise. Cloning mouse CD8+ T lymphocytes that will be first consider is to some extend facilitated by our complete control of the in vivo priming process an...
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Evaluating CD8+ T Cell Responses In Vitro
The 51Cr-release assay described in the 1960s has been for decades the gold standard cytolytic assay and remains in use in many laboratories. Whereas other radioactive tests were later on described, they never fully replaced the 51Cr-release assay. More thorough understanding of CTL biology and killing pathways has more recently resulted in the design of reliable nonradioactive tests to analyze CD8+ T cell responses which are likely to supplant in a close future the 51Cr-release assay. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Purification of Large Cytosolic Proteases for In Vitro Assays: 2S and 26S Proteasomes
Proteasomes are the main cytosolic proteases responsible for generating peptides for antigen processing and presentation in the MHC (major histocompatibility complex) class-I pathway. Purified 20S and 26S proteasomes have been widely used to study both specificity and efficiency of antigen processing. Here, we describe the purification of active human 20S and 26S proteasomes from human erythrocytes by DEAE-ion exchange chromatography, ammonium sulfate precipitation, glycerol density gradient centrifugation, and Superose-6 size exclusion chromatography and their characterization using fluorogenic substrates and specific inh...
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Bioinformatics Identification of Antigenic Peptide: Predicting the Specificity of Major MHC Class I and II Pathway Players
Bioinformatics methods for immunology have become increasingly used over the last decade and now form an integrated part of most epitope discovery projects. This wide usage has led to the confusion of defining which of the many methods to use for what problems. In this chapter, an overview is given focusing on the suite of tools developed at the Technical University of Denmark. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Recombinant Poxviruses: Versatile Tools for Immunological Assays
The study of antigen processing and presentation is critical to our understanding of the mechanisms that govern immune surveillance. A typical requirement of assays designed to examine antigen processing and presentation is the de novo biosynthesis of a model antigen. Historically, Vaccinia virus (VACV), a poxvirus closely related to Cowpox, has enjoyed widespread use for this purpose. Recombinant poxvirus-based expression has a number of advantages over other systems. Poxviruses accommodate the insertion of large pieces of recombinant DNA into their genome, and recombination and selection are relatively efficient. Poxviru...
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Purification, Preparation, and Use of Chaperone–Peptide Complexes for Tumor Immunotherapy
We describe a method for purifying Hsp70–peptide complexes that can be used to prepare molecular chaperone-based vaccines, involving sequential gel filtration, ion exchange, and affinity chromatography (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Identifying Source Proteins for MHC Class I-Presented Peptides
Identification of antigenic peptides recognized by cytolytic T lymphocytes (CTL) is a prerequisite for the development of targeted cancer immunotherapy approaches. This chapter provides a global approach for the identification of peptides recognized by CTL. It implies the identification of the HLA molecule presenting the peptide as well as the design and screening of a cDNA library derived from the tumor cells. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Biochemical Analysis of Naturally Processed Antigenic Peptides Presented by MHC Class I Molecules
Immune surveillance of infected or tumor cells by CD8+ T cells requires that MHC class I molecules present a diverse repertoire of peptides on the cell surface. Even a few copies of individual peptides among this mixture are sufficient for recognition by the antigen receptors of appropriate CD8+ T cells. Here we describe a method for biochemical analysis of the naturally processed peptides in living cells. The peptide mixture in cell extracts is fractionated using reverse phase high performance liquid chromatography and detected by the activation of CD8+ T cell hybridomas. The results provide information on the structure a...
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Quantitating MHC Class I Ligand Production and Presentation Using TCR-Like Antibodies
Accurately determining the number of peptide–MHC class I complexes on the cell surface is necessary when evaluating cellular processes or pharmaceuticals that alter the antigen presentation machinery. Here I describe a quantitative flow cytometry application for determining the number of peptide–MHC complexes on the surface of cells grown in tissue culture that express an endogenous protein from which the peptide is derived. The procedure requires a monoclonal antibody with the ability to distinguish MHC class I molecules presenting the peptide of interest from other peptide–MHC complexes. Fluorescence si...
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
