Neurite outgrowth measurement

Neurite outgrowth was measured as described previously (Hirose et al., 1998; Sandvig et al., 2004). To detect inhibition of ROCK, the dissociated cell clusters were incubated with proliferation-inducing medium containing 10 μM Y-27632 or an equivalent volume of sterile H₂O and then plated onto 10 μg/mL laminin (Sigma-Aldrich) for 1–2 hours. Cells treated with Y-27632 were spread onto the myelin substrate. Neurite outgrowth was quantified using ImageJ software (NIH, Bethesda, MD, USA).