Isolation and Culture of OPCs from Mouse Forebrain

OPCs were isolated from mixed male and female P5-8 Adgrg1^+/+ or Adgrg1^-/- mouse forebrains as previously described (Watkins et al., 2008 (link); Wang et al., 2001 (link)). Briefly, OPCs were purified by negatively selecting with mouse anti-Thy1.2 (BioRad, Hercules, CA; Cat #MCA02R) and mouse anti-GalC (Millipore, Burlington, MA; Cat #MAB342), followed by mouse anti-O4 (O4 hybridoma supernatant) for positive selection. After releasing OPCs from the O4 plate by trypsinization, cells were resuspended in proliferation media containing PDGF-AA and NT-3 (PreproTech, Rocky Hill, NJ ). OPCs were plated on coverslips coated with poly-D-lysine before coating with laminin I (R and D Systems, Minneapolis, MN; Cat#3400-010-01) or fibronectin (Millipore, Burlington, MA; Cat#341668) as previously described (Dugas et al., 2006 (link)). After 24 hr, rTG2 (2 nM) was added to the cultures and incubated for an additional 24 hr before fixation with 4% PFA and staining for PDGFRα and Ki67.