Open AccessApplication of confocal laser scanning microscopy to analysis of H 2 O 2 ‐induced DNA damage in human cells
Author(s) -
Fairbairn Daryl,
O'Neill Kim L.,
Standing Michael D.
Publication year - 1993
Publication title -
scanning
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.359
H-Index - 47
eISSN - 1932-8745
pISSN - 0161-0457
DOI - 10.1002/sca.4950150305
Subject(s) - dna , lysis , hydrogen peroxide , dna damage , confocal , agarose , confocal microscopy , agarose gel electrophoresis , microbiology and biotechnology , confocal laser scanning microscopy , biophysics , gel electrophoresis , chemistry , microscopy , single cell analysis , materials science , cell , biology , biochemistry , optics , physics
Confocal laser scanning microscopy (CLSM) offers improved depth discrimination and spatial resolution to the analysis of biologic samples. We demonstrate in this paper that such technology is valuable in examining DNA single‐strand breaks in human cells. The single‐cell‐gel (SCG) assay is a new technique for measuring DNA strand breaks in individual cells. Cells embedded in lowmelting‐point agarose are treated with varying concentrations of hydrogen peroxide to induce DNA strand breaks. Following cell lysis and alkaline electrophoresis, which enables single‐stranded break detection, analysis of the resulting “comets” provides an accurate method of comparing changes in DNA migration patterns, which have been shown to reflect the DNA damage levels. A statistically significant difference (p < 0.01) in single‐stranded DNA damage levels was detected in cells exposed to hydrogen peroxide concentrations as low as 10 nm for 2 min. LSM analysis of the SCG technique allows rapid, sensitive and reproducible quantitation of single‐stranded breaks of cellular DNA.
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