Isolation and Analysis of Mouse OPCs

Mouse primary oligodendrocyte progenitor cells (OPCs) were isolated from mixed glial cultures derived from postnatal day 1 mice as previously described [65 (link)]. Briefly, primary mixed glial cultures were established from the forebrains of postnatal C57BL/6 mice, and grown in media containing DMEM, 2 mM Glutamax, 1 mM sodium pyruvate, 20 mM HEPES and 10% fetal bovine serum. After 10 days, the flasks were shaken overnight to remove the OPCs [66 (link)], and OPCs were seeded on 12 mm glass cover slips coated with poly-L-lysine. At 12 h post-purification cultures, BubR1 expression in OPCs was determined by NG2/BubR1 or Olig2/BubR1 double staining. The following primary antibodies were used: anti-NG2 (Millipore, Rabbit, AB5320), anti-Olig2 (Millipore, Rabbit, AB9610), and anti-BubR1 (BD, Mouse, 612503).