As previously stated, each pathogen may bring with it its own set of specific requirements. These often become most apparent when a pathogen is transfected to express a normally soluble 'reporter' antigen (e.g. ovalbumin) and then processing of the same epitopes are compared after these two modes of delivery (17, 19}.
Soluble protein antigens can be readily titrated into APC assays within a concentration range from nanogram to milligram, normally without discernible adverse effects. Such titrations usually produce bell-shaped clones, with high dose inhibition usually attributed to complex immunoregulatory events. In contrast, adherent macrophage monolayers have a relatively narrow capacity to ingest particulate matter. At high multiplicity of infection, it may be possible to obtain 30-50 protozoa or bacteria per macrophage with greater than 95% infection levels. However, unlike soluble antigens, dose response characteristics may be
• 96-well flat-bottom tissue culture plates • (Nunc) .
• '6 + 2' BMM0 (see Protocol I) loaded with specific or irrelevant antigen by osmotic lysis (see Protocol 5) ,
• Complete tissue culture medium: supplemented DMEM plus 10% HI-FCS (see influenced by numerous complex or trivial factors. As MOl is reduced, both the number of organisms per macrophage (the true parameter to be evaluated) and the percentage of macrophages infected (a confounding variable) decrease. At MOls of 10:1, it is not uncommon to observe < 50% infection levels within a macrophage monolayer. Hence, under these conditions, apparent processing efficiency (as measured in a functional T cell assay) becomes a function of both actual processing efficiency and also presenting cell number (and, as a result, MHC density). Varying the MOI may also pose inherent problems at the top end of the dose response. Protocols J and 2 are based on antigen pulsing into macrophage monolayers, following by exhaustive removal of excess antigen. At high MOI, the adherence properties of macrophages may vary considerably, and after washing of such monolayers, a rather patchy distribution of macrophages with lower than expected MOI may remain. Furthermore, the ability to effectively remove particulate organisms from 96-well plates is rather poor at high MOI, increasing the chance of antigen uptake and hence new processing occurring over an extended time period. In practice, therefore, in routine pulsing experiments, the effective range of infection levels which can be tested is rather small. A variation of the antigen pulsing protocols we have found useful in extending the effective assay range, involves infection of non-adherent BMM0 in 'non-stick' polypropylene tubes and is given in Protocol 7. Various safety considerations may need to be introduced, e.g. to reduce aerosols or the volume of biohazardous waste material.
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