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

Glial cells were separated from myelin and red blood cells using a 30% Percoll gradient (Hirayama et al., 1983; Kim et al., 1985) . The gradient was centrifuged in an ultracentrifuge (Beckman Instruments, Palo Alto, CA) equipped with a JA20 fixed-angle rotor spun at 14,000 rpm for 45 min at 4°C. The glial cell layer, the region below the myelin cap and above the red blood cells, was transferred to a 50-ml tube that was then filled with isolation medium and spun for 10 minutes at 1,500 rpm in a centrifuge (GLC-2B; Sorvall Div.). The supernatant was removed and the pelleted cells were resuspended (800 pl/spinal cord) in 10% FBS in DME with 584 mg/liter L-glutamine, 4.5 g/liter v-glucose, 25 #g/ml gentamycin, and 1 mM sodium pyruvate. In all cases, 200 ~tl of cell suspension was plated per 35-mm plastic dish (coated with extracellular matrix; Accurate Chemical & Scientific Corp., Westbury, NY). After incubating for 1 h at 37°C to enhance attachment, the cultures were fed with 2 ml of 10% FBS-DME solution. At 1 and 3 d in vitro (die) cultures were fed with 0.5 % FBS in defined medium (DME with 584 mg/liter L-glutamine, 4.5 g/liter v-glucose, 5/~g/ml insulin, 50 /~g/ml transferrin, 30 nM selenium, 30 nM triiodothyronine, 25 #g/ml gentamycin, and 1 mM sodium pyruvate; modified from Eccleston and Silberberg, 1984) . The cultures were grown in this low serum defined medium to enhance the growth of oligodendrocytes relative to fibroblasts. After l, 3, or 6 die, cultures were fixed with 2% paraformaldehyde in MEM-Hepes for 15 min.