By Solheim J.C.
Well-recognized and cutting edge experimentalists element their state-of-the-art tools for learning the antigen processing and presentation. Drawing on services from biochemistry, phone biology, and immunology, they describe step by step tools designed to query how MHC-binding peptides are generated, to check how peptides are dropped at MHC molecules, to investigate MHC peptide-binding styles, and to assay the T-cell reaction to the MHC/peptide complicated. Emphasis is given these technical steps serious for experimental good fortune which are usually passed over from tools 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 functional instruments that might expand the questions that may be requested, and successfully be spoke back, bearing on antigen processing/presentation.
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Additional info for Antigen Processing and Presentation Protocols (Methods in Molecular Biology Vol 156)
If the anticlass I Ab is not directly labeled, a FITC-conjugated anti-immunoglobulin Ab, specific for the anticlass I Ab. 5. Ethidium homodimer (Molecular Probes, Eugene, OR). Stock solution in PBS–BSA (100 µg/mL). 6. Flow cytometer. 7. 3. 6. 1. Infection of Target Cells with VV 1. 3. 2. rVV viruses expressing the proteins of interest. Controls, including a wild type VV, or an irrelevant rVV, would be included as well. 3. BFA stock (25 mg/mL) in methanol. 4. 5%). 5. Na51CrO4, 10 mCi/mL. Proteasome Inhibitors 23 6.
Incubate for 1 h at 37°C. 5. Add 5 mL culture medium and pellet cells. Aspirate as much media as possible, resuspend cells in warm medium (with BFA) and incubate for 10–15 min at 37°C. Pellet, and again scrupulously remove the supernatant. Finally, resuspend the labeled cells in medium, containing BFA, at a density of 105 cells/mL. 6. Add 100 µL/well (104 cells) of the cell suspension in round-bottom 96-well plates. These may contain different numbers of the peptide-specific CTL, in 100 µL medium, in order to have different effector-to-target ratios.
2. 1. FITC Labeling of Ag It is essential to demonstrate internalization of Ag before degradation and peptide loading of MHC-I. Studies with inhibitors may contribute evidence that Ag is internalized, but only measurement of uptake can provide clear details of the rate of endocytosis and of the effectiveness of inhibitors. To perform such analysis, it is necessary to label Ag with a marker enabling measurement of endocytosis. A number of possible ways of labeling proteins exist, such as iodination or biotinylation, but labeling with fluorophores, particularly FITC, is probably the easiest.
Antigen Processing and Presentation Protocols (Methods in Molecular Biology Vol 156) by Solheim J.C.