Differential tumor necrosis factor alpha expression by astrocytes from experimental allergic encephalomyelitis-susceptible and -resistant rat strains

Primary Glial Cell Cultures. Primary glial cell cultures were established from neonatal rat cerebra as previously described (31) . Meninges were removed before culture. Culture medium was Dulbecco's modified essential medium (DMEM), high glucose formula supplemented with glucose to a final concentration of 6 g/liter, 2 mM glutamine, 0.1 mM nonessential amino acid mixture, 0.1% gentamycin, and 10% FCS (Hyclone Laboratories, Logan, UT) . Oligodendrocytes were separated from the astrocytes by mechanical dislodging after 14 d in primary culture, and then the astrocytes were obtained by trypsinization (0.25% trypsin, 0.02% EDTA) . The astrocytes were monitored for purity by immunofluorescence. The cells were stained for glial fibrillary acidic protein (GFAP), an intracellular antigen unique to astrorytes, using a mAb to GFAP (1 :4) for 30 min at room temperature, followed by a 30-min incubation with goat anti-mouse Ig-FITC (1:20). Astrocyte cultures were routinely >97% positive for GFAP, and <2% of the cells were microglia based on their positive staining for nonspecific esterase and MAC-1, a mAb that reacts with the C3b receptor. In subsequent experiments, astrocytes were purified by four repetitions of trypsinization and replating to remove contaminating microglia ; after such manipulation, the astrocyte cultures were >99% positive for GFAP, and negative for nonspecific esterase and MAC-1 staining. Microglia were purified by a differential adhesion technique as described by Sasaki et al. (32) . Confluent mixed glial cultures were shaken at 270 rpm for 3 h, at which time floating cells were removed. The cells were plated in a 25-cmz tissue culture flask, and allowed to adhere for 1 h. Microglia adhere to plastic during this time period, while contaminating astrocytes and oligodendrocytes remain in the media. The adherent cells were positive for MAC-1 and nonspecific esterase (90%).