Measurement of DNA damage in mammalian cells using flow cytometry

A. E. Milner, Andrew T M Vaughan, I. P. Clark

Research output: Contribution to journalArticle

35 Scopus citations

Abstract

A technique for the detection of DNA damage induced by radiation insult has been developed. Cells were lysed with a buffer containing 2 M sodium chloride to release the DNA in a supercoiled from, the nucleoid. These were stained with the DNA intercalating dye, ethidium bromide, and exposed to laser light within a flow cytometer. Scattered and fluorescent light was analyzed form the laser/nucleoid interaction following irradiation of viable cells with γ rays. The addition of ethidium bromide to prepared nucleoids caused a reduction in scattered light due to condensation of the nucleoid. Irradiation of cells prior to nucleoid production and ethidium bromide treatment restricted this condensation and produced a dose-dependent increase in laser scatter. Nucleoids derived from human lymphocytes showed enhanced light scatter from 5 Gy, compared to Chinese hamster ovary (CHO) fibroblasts where doses above 10 Gy were required. Up to 30 Gy CHO nucleoids showed a dose-dependent reduction in the ethidium bromide fluorescence. This technique allows detection of altered light scattering and fluorescent behavior of nucleoids after cellular irradiation; these may be related to structural changes within the nucleus induced by the radiation. The use of flow cytometry compared to other methods allows a rapid analysis of nuclear damage within individual cells.

Original languageEnglish (US)
Pages (from-to)108-117
Number of pages10
JournalRadiation Research
Volume110
Issue number1
StatePublished - 1987
Externally publishedYes

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ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biophysics
  • Radiology Nuclear Medicine and imaging
  • Radiation

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