Coronavirus induction of class I major histocompatibility complex expression in murine astrocytes is virus strain specific

RtA. The expression of class I molecules was measured in astrocytes cultured in flat-bottomed 96-well plates at a density of 10 4 cells/well and infected with various virus strains at a multiplicity of infection (m.o.i.) of 1-2. Mock infected cells served as controls. On day 3 or 5 p.i., cells were washed twice using a wash buffer of 0.3% BSA in 0.1 M PBS before the addition of 50/A of the appropriate mAbs in triplicate. After a 60 rain incubation at room temperature, cells were washed three times, followed by the addition of 20,000 cpm/well of 12SI-labeled protein A (30 /~Ci//zg; ICN Biomedicals, Costa Mesa, CA). At the end of a second 60min incubation, cells were extensively washed in PBS to remove unbound radiolabeled protein A and detached with 0.5% trypsin-0.2% EDTA (JRH Biosciences). Data are presented as cpm bound radioactivity + SD, or percent increase in class I expression in infected cells compared with that in uninfected controls, corrected for background binding in the absence of antibody. A result was considered positive when the percent increase in expression was 100% or greater. Nonspecific staining was identified by the inclusion of Kd-specific mAb SF1-1.1.1. In some experiments, supernatants from infected cells were substituted for virus and RIA performed to detect class I 3 d later.