Open AccessOptimization of Single-Cell Gel Electrophoresis (SCGE) for Quantitative Analysis of Neuronal DNA Damage
Author(s) -
Erick J. Morris,
J.C. Dreixler,
Ke-Yi Cheng,
Peter M. Wilson,
R.M. Gin,
Herbert M. Geller
Publication year - 1999
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/99262st02
Subject(s) - dna damage , gel electrophoresis , comet assay , dna , biology , dna repair , microbiology and biotechnology , apoptosis , biochemistry
Neuronal death can be induced by DNAdamaging agents and occurs by apoptosis involving a specific signal-transduction pathway. However, to our knowledge, methods for the quantitative determination of DNA damage in individual neurons have not yet been described. Here we optimize the single-cell gel electrophoresis (SCGE) or “comet”-assay to measure DNA damage within individual neurons growing in dissociated cell culture. In addition, we have written a macro for the NIH Image program to determine the tail moment of individual comets. We have calibrated this method using γ-irradiated (0–16 Gy) cerebral cortical neurons from the rat central nervous system. Neuronal DNA damage (in the form of DNA strand breaks) occurs in a linear, dose-dependent manner, which can be quantitatively determined in vitro using the SCGE assay. These data demonstrate that the SCGE assay is an effective method to measure DNA damage in individual neurons and may be highly useful for the study of neuronal DNA damage formation, repair and apoptosis.