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

Primary Astrocyte Cultures. Astrocytes were isolated from mixed glial cell cultures prepared from the brains of newborn C57B1/6 mice (Bantin and Kingman, Fremont, CA) at postnatal day 0-3 according to McCarthy and deVellis (48) . Briefly, single cell suspensions were prepared from cerebri dissected free of brain stems and cerebelli, plated at 3-5 brains per T-75 flask and allowed to grow to confluence at 12-15 d in vitro. Culture medium consisted of DMEM/Ham's F12 (1:1; JRH Biosciences, Lenexa, KS) supplemented with 10% FCS (Gemini Bioproducts, Inc., Calabasas, CA), 15 mM Hepes, 2.5 mM t-glutamine, and penicillin/streptomycin (100 U/ml-100 ~g/ml). At confluence the cultures were mechanically shaken to dislodge microglia and oligodendroglia, resulting in preparations enriched 95% or greater for cells staining for glial fibrillary acidic protein (GFAP). Immunoperoxidase or immunofluorescent staining (described below) revealed that the cell preparations contained ,-2-4% of cells of microglia/macrophage lineage, expressing F4/80, T-200, the mouse equivalent of human common leukocyte antigen (CD45), and/or Mac-1 surface markers.