Preparation of stable single-chain trimers engineered with peptide, beta2 microglobulin, and MHC heavy chain.
Authors: Hansen T, Yu YY, Fremont DH
Abstract
This unit describes a method for constructing a class I MHC molecule with a bound peptide as a single polypeptide chain, termed SCT, for single chain trimer. The component organization of the SCT appears to be widely applicable to different mouse or human MHC class I isotypes bound by different antigenic peptides. The enhanced peptide occupancy afforded by the SCT format makes these molecules effective reagents as DNA vaccines, multimeric staining reagents to enumerate CD8 T cells, and probes of lymphocyte biology.
PMID: 19918946 [PubMed - indexed for M...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Generation of peptide MHC class I monomers and multimers through ligand exchange.
Authors: Toebes M, Rodenko B, Ovaa H, Schumacher TN
Abstract
The recognition of defined antigen-MHC complexes by antigen-specific T cells forms the molecular basis of T cell immunity. It has been shown that fluorescently labeled recombinant MHC tetramers can be utilized to detect antigen-specific T cells by flow cytometry. Since this first description, MHC tetramers and other types of MHC multimers have become a core tool to monitor the development of disease- and therapy-induced antigen-specific T cell responses both in humans and in animal model systems. This unit describes a set of protocols that transf...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Visualization of multiprotein complexes by flow cytometry.
Authors: Schrum AG
Abstract
Multiprotein complexes and other protein-protein interactions play important roles in virtually all cellular processes. Analysis of coimmunoprecipitation of protein complexes by flow cytometry (IP-FCM, or "the fly-p" method) provides a sensitive means to measure these interactions in the native/nondenatured state. First, immunoprecipitating antibodies are covalently coupled to polystyrene latex beads whose low autofluorescence is compatible with flow cytometry. These antibody-coupled beads are used to immunoprecipitate a specific protein (primary analyte) present in cell lysates...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Monocyte and neutrophil isolation and migration assays.
Authors: Yona S, Hayhoe R, Avraham-Davidi I
Abstract
This unit describes methods for isolating mouse monocytes and neutrophils, as well as in vitro protocols for measuring cell migration and polarization. The method employed here for the isolation of naïve phagocytes overcomes many of the difficulties previously encountered concerning phagocyte activation. Three in vitro protocols are provided for the analysis of cell migration, one requiring no specialized equipment, one requiring the modified Boyden chamber, and the other employing a flow chamber, which measures cell adhesion, rolling, and migration. Fi...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Experimental autoimmune encephalomyelitis in the mouse.
Authors: Miller SD, Karpus WJ, Davidson TS
Abstract
This unit details the materials and methods required for both active induction and adoptive transfer of experimental autoimmune encephalomyelitis (EAE) in the SJL mouse strain using intact proteins or peptides from the two major myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). Detailed materials and methods required for the purification of both PLP and MBP are also described. A protocol for isolating CNS-infiltrating lymphocytes in EAE mice is included. Modifications of the specified protocols may be necessary for efficient induc...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Choosing and preparing antigen-presenting cells.
Authors: Harding CV, Canaday D, Ramachandra L
Abstract
The first issue in selecting a system for antigen-presentation experiments is to define the appropriate type of antigen-presenting cell (APC) to study. For some experiments, crude preparations such as splenocytes or peripheral blood mononuclear cells (PBMCs) may suffice to provide APC function for stimulating T cells. This unit develops approaches for preparation of more defined APC populations, including dendritic cells (DCs), macrophages, and B lymphocytes, the three types of "professional" APC. Each of these cell types exists in different stages of ...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Presenting exogenous antigen to T cells.
Authors: Harding CV, Ramachandra L
Abstract
Antigen processing and presentation experiments can be done with a wide variety of antigen-presenting cells (APCs). Most experiments will use one of the "professional" APC types: dendritic cells (DCs), macrophages, and B lymphocytes. Other types of cells may be used for antigen presentation in some circumstances. Each type of professional APC has an important antigen-presentation function, but the different APC types contribute to different aspects of the immune response. Therefore, selection of an APC type for study must include consideration of the stage or asp...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
The MHC motif viewer: a visualization tool for MHC binding motifs.
Authors: Rapin N, Hoof I, Lund O, Nielsen M
Abstract
In vertebrates, the onset of cellular immune reactions is controlled by presentation of peptides in complex with major histocompatibility complex (MHC) molecules to T cell receptors. In humans, MHCs are called human leukocyte antigens (HLAs). Different MHC molecules present different subsets of peptides, and knowledge of their binding specificities is important for understanding differences in the immune response between individuals. Algorithms predicting which peptides bind a given MHC molecule have recently been developed with high prediction accuracy....
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Use of the hemagglutinating virus of Japan (HVJ) envelope as a versatile delivery system for nucleic acids and proteins to leukocytes in vitro.
Authors: Kondo Y, Miyata K, Kato F
Abstract
The hemagglutinating virus of Japan (HVJ; also called the Sendai virus) envelope has been developed as a safe and efficient non-viral vector. Because replication and transcription of genomic RNA is inactivated by beta-propiolactone treatment or UV-irradiation, the HVJ envelope is extremely safe. This unit describes the method of transfection of siRNA and protein with the HVJ envelope.
PMID: 20376840 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Detecting tyrosine-phosphorylated proteins by Western blot analysis.
Authors: Sawasdikosol S
Abstract
The development of monoclonal antibodies (mAbs) that recognize nearly all of the phosphorylated tyrosine residues, irrespective of the surrounding sequences, enables researchers to detect the phosphorylation state of proteins through the use of anti-phosphotyrosine western blotting. The availability of this simple, reliable, nonradioactive and yet sensitive method created a boom in signal transduction research. While the methodology of how to perform an anti-phosphotyrosine western blot remains unchanged since the procedure became widely used in the early part of 1990s, ste...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Collagen-induced arthritis.
Authors: Rosloniec EF, Cremer M, Kang AH, Myers LK, Brand DD
Abstract
The mouse model collagen-induced arthritis (CIA) is a widely studied autoimmune model of rheumatoid arthritis. In this model, autoimmune arthritis is induced by immunization with type II collagen (CII) emulsified in complete Freund's adjuvant. This unit describes the steps necessary for the acquisition, handling, and preparation of CII, in addition to the selection of mouse strains, proper immunization technique, and methods for evaluation of the incidence and severity of arthritis. In this model, the first signs of arthritis appear appr...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Efficient delivery of antibody into living cells using hemagglutinating virus of Japan (HVJ) envelope.
Authors: Kondo Y, Miyata K, Kato F
Abstract
This unit describes a novel method for antibody delivery into living cells using HVJ (hemagglutinating virus of Japan) envelope, an inactivated Sendai virus particle.
PMID: 20376843 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Endoglycosidase and glycoamidase release of N-linked glycans.
Authors: Freeze HH, Kranz C
Abstract
Nearly all proteins entering the lumen of the endoplasmic reticulum (ER) become glycosylated en route to a cellular organelle, the plasma membrane, or the extracellular space. Many glycans can be attached to proteins, but the most common are the N-linked glycans (oligosaccharides). These chains are added very soon after a protein enters the ER, but they undergo extensive remodeling (processing), especially in the Golgi. Processing changes the sensitivity of the N-glycan to enzymes that cleave entire sugar chains or individual monosaccharides, which also changes the migr...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Signal transduction during activation and inhibition of natural killer cells.
Authors: Watzl C, Long EO
Abstract
Natural killer (NK) cells are important for early immune responses to viral infections and cancer. Upon activation, NK cells secrete cytokines and chemokines, and kill sensitive target cells by releasing the content of cytolytic granules. This unit is focused on the signal transduction pathways that regulate NK cell activities in response to contact with other cells. We will highlight signals regulating NK cell adhesion to target cells and describe the induction of cellular cytotoxicity by the engagement of different NK cell activation receptors. Negative signaling induce...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation and functional use of human NKT cells.
Authors: Exley MA, Wilson B, Balk SP
Abstract
This unit details methods for the isolation, in vitro expansion, and functional characterization of human iNKT cells. The term iNKT derives from the fact that a large fraction of murine NKT cells recognize the MHC class I-like CD1d protein, are CD4+ or CD4-CD8- (double negative), and use an identical "invariant" TCRalpha chain, which is generated by precise Valpha14 and Jalpha281 (now renamed Jalpha18) rearrangements with either no N-region diversity or subsequent trimming to nearly identical amino-acid sequence (hence, 'iNKT'). Basic Protocol 1 and Alternate P...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
