A rapid method to measure beta-amyloid induced neurotoxicity in vitro.
Beta-amyloid (Abeta) is the primary protein component of senile plaques in Alzheimer's disease (AD) and is believed to be associated with neurotoxicity in the disease. Abeta-induced neurotoxicity is strongly dependent on its structure. The aggregation of the peptide from monomeric form to fibrils is a function of many variables including time. It is because of this dynamic nature of Abeta structure that there is a necessity for an in vitro toxicity assay that is rapid enough to eliminate or at least lower the possibility of Abeta structural changes during the time required for the assay. Here we describe a fast and sensitive method with which to assess Abeta-induced neurotoxicity in SH-SY5Y neuroblastoma cells. The method employs two-color flow cytometry using annexin-phycoerythrin, a marker for cell surface phosphatidylserine that typically indicates early stages of apoptosis, and 7-amino-actinomycin D, a membrane impermeant nucleic acid dye. We compare results using the two-color assay to those obtained using the propidium iodide toxicity assay and demonstrate comparability of results, but in 2h or less with the two-color assay as opposed to 24-48 h with the propidium iodide assay. The assay described could be a useful tool in evaluating the role of Abeta structure in biological activity of the peptide.
Patel
Dhara
D
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA. dhara1@umbc.edu
Good
Theresa
T
D016229:Amyloid beta-Peptides / Q000032:analysis* / Q000633:toxicity*; D000704:Analysis of Variance; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003609:Dactinomycin / Q000031:analogs & derivatives; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D009447:Neuroblastoma / Q000473:pathology; D011419:Propidium; D013997:Time Factors
D016229:Amyloid beta-Peptides; D003609:Dactinomycin; D011419:Propidium; C025942:7-aminoactinomycin D