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Journal of Histochemistry and Cytochemistry, Vol. 47, 661-672, May 1999, Copyright © 1999, The Histochemical Society, Inc.

ARTICLE

Direct Temporal Analysis of Apoptosis Induction in Living Adherent Neurons

Andrea M. Vincenta and Kenneth Maiesea
a Laboratory of Cellular and Molecular Cerebral Ischemia, Departments of Neurology and Anatomy and Cell Biology, Center for Molecular and Cellular Toxicology and Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan

Correspondence to: Kenneth Maiese, Neurology and Anatomy & Cell Biology, 6E-19 UHC, Wayne State U. School of Medicine, 4201 St. Antoine, Detroit, MI 48201.

Destruction of neurons through the genetically directed process of programmed cell death (PCD) is an area of intense interest because this is the underlying mechanism in a variety of developmental and neurodegenerative diseases. The ability to identify and track viable neurons subjected to PCD could be invaluable in development of strategies to prevent or reverse the downstream mechanisms of neuronal PCD. We have developed a novel assay for PCD in viable, adherent cells using annexin V labeling. Annexin V binds to the highly negatively charged plasma membrane phosphatidylserine residues that undergo membrane translocation during PCD. Current annexin V techniques are almost exclusively restricted to flow cytometric analysis. Our unique technique permits repeated examination of individual viable neurons without altering their survival. Correlation with electron microscopy and dye exclusion assays demonstrate both sensitivity and specificity for our method to detect PCD. To our knowledge, this is the first account of a technique that positively identifies PCD in viable, adherent cells. (J Histochem Cytochem 47:661–671, 1999)

Key Words: annexin V, apoptosis, fluorescence microscopy, neurodegeneration, nitric oxide, phosphatidylserine residues, primary hippocampal neurons, rat


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