The PC12 cell line is a widely used in vitro model for screening the neuroprotective activity of small molecule libraries. External insult due to serum deprivation or addition of etoposide induces cell death by apoptosis. While this screening method is commonly used in early stage drug discovery no protocol accounting for cell passage number effect on neuroprotective activity has been disclosed. We herein report that passage variation results in false-positive/false-negative identification of neuroprotective compounds; undifferentiated PC12 cells with high passage number are less sensitive to injury induced by serum-deprivation or etoposide treatment. In contrast, NGF differentiated PC12 cells of later passage number are more sensitive to injury induced by etoposide than lower passage number but only after 72 h. Passage number also affects the adherence phenotype of the PC12 cells, complicating screening assays. We report an optimized protocol for screening the neuroprotective activity of small molecules in PC12 cells, which accounts for passage number variations.
Keywords: PC12 cells; apoptosis; cell viability; neuroprotection assay; protocol; screening.