title: Passage Variation of PC12 Cells Results in Inconsistent Susceptibility to Externally Induced Apoptosis.
abstract: 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.
authors.forename: Nihar
authors.initials: N
authors.lastname: Kinarivala
authors.forename: Kaushik
authors.initials: K
authors.lastname: Shah
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Abbruscato
authors.affiliation: Center for Chemical Biology, Department of Chemistry and Biochemistry, Texas Tech University , Lubbock, Texas 79409, United States.
authors.forename: Paul C
authors.initials: PC
authors.lastname: Trippier
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002454:Cell Differentiation / Q000187:drug effects; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005047:Etoposide / Q000494:pharmacology; D018696:Neuroprotective Agents / Q000494:pharmacology*; D016716:PC12 Cells; D051381:Rats; D013997:Time Factors; D059005:Topoisomerase II Inhibitors / Q000494:pharmacology
chemical_list: D016895:Culture Media, Serum-Free; D018696:Neuroprotective Agents; D059005:Topoisomerase II Inhibitors; D005047:Etoposide
keywords: PC12 cells; apoptosis; cell viability; neuroprotection assay; protocol; screening