Neurite outgrowth assay

PC12 cells (1 × 10⁴ cells/well) were plated on a 24-well tissue culture plate pre-coated with collagen type IV (Corning) containing a complete culture medium. After 24 h, the medium was replaced with serum free DMEM containing 100 ng/mL recombinant rat beta-NGF (R&D systems, Cat # 556-NG, Minneapolis, MN, USA), N2 supplement (Thermo Fisher Scientific, Cat # 17502048) and 0.5 % FBS to induce neurite growth in the presence or absence of oxaliplatin (200 nM, LC Laboratories, Woburn, MA, USA). WLR (25 and 100 μg/mL) or amifostine (0.5 mM, Santa Cruz Biotechnology, Dallas, TX, USA) were co-treated with oxaliplatin. Three days after the drug treatment, a morphometric analysis was done on digitalized images of PC12 cells taken with an inverted microscope (IX71, Olympus, Center Valley, PA, USA). Images of fields with more than 20 cells were captured and used for the image analysis. The differentiation of cells induced by NGF was examined, and cells that had at least one neurite with a length equal to the cell body diameter were counted. The number of neurite bearing cells was expressed as a percentage of the total cells. Total neurite length was determined by manually tracing the length of the neurite per cell using the MetaMorph image software (Molecular Devices).