Optimal conditions of single cell gel electrophoresis(comet) assay to detect DNA single strand breaks in mouse lymphoma L5178Y cells.

Title
Optimal conditions of single cell gel electrophoresis(comet) assay to detect DNA single strand breaks in mouse lymphoma L5178Y cells.
Authors
류재천권오승Hyung-Tae Kim
Keywords
single cell gel electrophoresis; Comet; Tail Moment; Mouse lymphoma L5178 cell; DNA single strand breakage; Methyl Methanesulfonate; Benzo(a)pyrene
Issue Date
2001-12
Publisher
Environmental Mutagens & Carcinogens (한국환경성돌연변이발암원학회지)
Citation
VOL 21, NO 2, 89-94
Abstract
Recently, single cell gel electrophoresis, also known as comet assay, is widely used for the detection and measurement of DNA strand breaks in vitro and in vivo in many toxicological fields such as radiation exposure, human monitoring and toxicity evaluation. As well defined, comet assay is a sensitive, rapid and visual method for the detection of DNA strand breaks in individual cells. Briefly, a small number of damaged cells suspended in a thin agarose gel on a microscope slide were lysed, unwinded, electrophoresed, and stained with a fluorescent DNA binding dye. The electric current pulled the charged DNA from the nucleus such that relaxed and broken DNA fragments migrated further. The resulting images which were subsequently named for their appearance as comets, were measured to determine the extent of DNA damages. However, some variations could be occurred in procedures, laboratories's conditions and kind of cells used. Hence, to overcome and to harmonize these matters in comet assay, International Workshop on Genotoxicity Test Procedure (IWGTP) was held with several topics including comet assay at Washington D.C. on March, 1999. In spite of some consensus in procedures and conditions in IWGTP, there are some problems still remained to be solved. In this respect, we attempted to set the practical optimal conditions in the experimental procedures such as lysis, unwinding, electrophoresis and neutralization conditions and so on. First of all, we determined optimal lysis and unwinding time by using 150 mM methyl methanesulfonate (MMS) which is usually used concentration. And then, we determined optimal positive control concentrations of benzo(a)pyrene (BaP) and MMS in the presence and absence of S9 metabolic activation system, respectively.
URI
http://pubs.kist.re.kr/handle/201004/16702
ISSN
1225-6307
Appears in Collections:
KIST Publication > Article
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