In vitro analysis of the oligodendrocyte lineage in mice during demyelination and remyelination

At 1, 3, 4, 5, or 8 wpi, mice were anesthetized with methoxyflurane and then decapitated. For each glial cell isolation, spinal cords remov&t from three infected mice exhibiting neurological dysfunction (Woyciechowska et al., 1984) were combined and spinal cord tissue from three age-matched control mice was prepared simultaneously. Spinal cords were minced and then dissociated according to a procedure modified from Miller et al. (1985) . The tissue was incubated at 37°C with enzymes (twice for 20 rain in 0.125% trypsin with 0.02% collagenase in MEM-Hepes followed by one 15-min incubation in 0.05 % trypsin with 0.53 M EDTA), bathed in a solution that inhibited trypsin and digested free DNA (0.25% soybean trypsin inhibitor, 0.002% DNase I, 0.166% BSA, and 5% FBS in DME) and passaged through pipettes (5 ml, 10 times; 1 ml, 10 times; Pasteur pipette 10 times). Floating cells were transferred to a 50-ml tube which was then filled with isolation medium (1 mM Hepes, 50 U/ml penicillin, 50 #g/ml strep~ tomycin, and 25/zg/ml gentamycin in HBSS without calcium or magnesium, pH 7.4) and spun for 10 min at 1,500 rpm in a centrifuge (GLC-2B; Sorvall Die., Newton, CT). The supernatant was removed and pelleted cells were resuspended in 10 ml of isolation medium.