By Jill R. Beyette, Timothy Hubbell (auth.), Joyce C. Solheim (eds.)
The processing and presentation of antigens by means of significant histocompatibility advanced (MHC) molecules is a vital immunological phenomenon in infectious illness, malignancy, autoimmune affliction, and transplantation. In Antigen Processing and Presentation Protocols, well-recognized and cutting edge experimentalists aspect their state of the art tools for learning this complicated method. Drawing on services from biochemistry, telephone biology, and immunology, they describe step by step equipment designed to question how MHC-binding peptides are generated, to check how peptides are dropped at MHC molecules, to research MHC peptide-binding styles, and to assay the T phone reaction to the MHC/peptide advanced. each one procedure is gifted in enough aspect to be without problems reproducible and contains notes approximately power pitfalls and how one can stay away from disasters. Emphasis is given these technical steps severe for experimental luck which are usually passed over from equipment released within the fundamental literature.
Eminently available and state of the art, Antigen Processing and Presentation Protocols presents either new and skilled investigators with hugely sensible instruments that would expand the questions that may be requested, and successfully be replied, relating antigen processing/presentation.
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Additional resources for Antigen Processing and Presentation Protocols
However, the results from the use of any one inhibitor, or group of inhibitors, cannot, in itself, implicate a particular route of trafficking. Rather, the consensus between the effects of inhibitors affecting widely differing processes is strongly suggestive of cytosolic or endosomal trafficking. Concentrations of stock and working solutions of inhibitors, and the lengths of time required for inhibitor efficacy, are shown in Table 2. 4. Notes 1. It is essential that the time of Ag pulsing is kept to a minimum.
Ten thousand events, or more, may be counted. The mean channel florescence values can be used to estimate the amount of class I expressed in the cell surface, with the background levels given by the cells incubated in the presence of BFA. 3. 1. INFECTION OF TARGET CELLS WITH RVV The method described here uses vaccinia as an expression vector, but it can be used with different viruses (see Note 9). BFA is added before 51Cr labeling, in order to avoid exposing CTLs to proteasome inhibitors. BFA prevents the presentation of peptides generated after inhibitor removal.
2. Strategies Once a suitable readout system has been established, one may proceed with investigating the trafficking of Ag and MHC-I molecules. Initially, it is imperative to rule out peptide contamination in the Ag preparation. Failure to do so will leave the possibility that peptide contaminants in the Ag preparation are binding to MHC-I directly, either at the cell surface or at another site. Once peptide contamination has been ruled out, a number of approaches can be used to study uptake of the Ag into the APC.