For both MHC class I- and class II-restricted antigen processing, three simple experimental parameters need to be described, namely dose dependency, kinetics, and sensitivity to various inhibitors which define the biochemical/cell biological route of processing. Complete protocols for the determination of these parameters for class I- and class II-restricted presentation of a simple protein antigen, e.g. ovalbumin, are given below.
3.1.1 Generation of murine bone marrow-derived macrophages We have had success in assays of class I and class II processing using bone marrow-derived macrophages. However, with minor modification, the subsequent protocols for addressing antigen processing should be applicable to any other monocyte/macrophage population cultured in vitro or harvested ex vivo. It should not be assumed that all macrophages will process similarly. Not only may kinctics of antigen uptake vary, but the vacuolar compartments and enzymatic machinery may be subtly different. Furthermore, it has now been realized that cytokine regulation of macrophage function extends to that of regulation of processing enzymes, introducing another major confounding variable should ex vivo macrophage populations be compared from inflammatory versus quiescent tissue sites. Protocol 1 describes one method for the derivation of bone marrow-derived macrophages for studies of antigen processing and presentation.
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