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

Immunoneutralization was also used to identify the possible presence of IFN in UV-irradiated supernatants of A59qnfected astrocytes. For this purpose, rat monoclonal anti-mouse IFN-3' antibody (hybridoma R4-6A2; ATCC HB 170) or rabbit polyclonal anti-mouse IFN-a/3 (Lee Biomolecular) was added to the astrocytes before the addition of A59, and class I expression measured by RIA on day 3 p.i. Anti-IFN-3' (20-60/zg/ml) completely inhibited the protective activity of 10 U/ml of recombinant mouse IFN-3', whereas 10-30 U/ml anti-IFN-a/r effective in neutralizing 20 U/ml purified mouse IFN-c~//~. Detection of TNE TNF activity was measured in a cytotoxicity assay using actinomycin D-treated L929 fibroblasts as targets, again with vital dye uptake as a spectrophotometric endpoint. Serial 2-fold dilutions of UV-irradiated supernatants, or 10-fold dilutions of murine recombinant TNF-ot (Genzyme Corp.), as a standard were added to L929 monolayers in 96-well plates in the presence of actinomycin D (8 #g/ml; Sigma Chemical Co., St. Louis, MO). After an 18-h incubation at 37~ 7% CO2, neutral red was added as described for the IFN assay, and OD read at 540 nm.