4.7. Neurite Outgrowth Assay in PC12

Neurite outgrowth assay in PC12 was performed as described previously [39]. Briefly, PC12 cells (1 × 10⁴ cells/well) were cultured on a collagen type IV-coated 24-well plate for 24 h. To induce neurite growth, the cells were replaced with differentiation medium consisting serum-free DMEM, 100 ng/mL recombinant rat beta NGF (R&D Systems, Minneapolis, MN, USA), 1% N2 supplement (Thermo Fisher Scientific) and 0.5% FBS for 4 days. To investigate the effect of LOHP on neural differentiation of the PC12 cells, the cells were exposed to DM containing a combination of LOHP (200 nM), EFVF, and each constituent or amifostine (0.5 mM) as described earlier. A neurite outgrowth analysis was performed using a phase contrast bright-field inverted microscope (IX71, Olympus, Tokyo, Japan). Digitalized morphometric images of each well were obtained and the fields containing more than 20 cells were captured. The cells bearing neurite that had at least one neurite with a length longer than the diameter of the cell body were counted from four captured images and expressed as a percentage of the total of 80 cells counted from the images. Total neurite lengths were measured by manually tracing the length of the neurites using the Meta Morph image software (Molecular Devices).