Optimal conditions of single cell gel electrophoresis(comet) assay to detect DNA single strand breaks in mouse lymphoma L5178Y cells.
- Optimal conditions of single cell gel electrophoresis(comet) assay to detect DNA single strand breaks in mouse lymphoma L5178Y cells.
- 류재천; 권오승; Hyung-Tae Kim
- single cell gel electrophoresis; Comet; Tail Moment; Mouse lymphoma L5178 cell; DNA single strand breakage; Methyl Methanesulfonate; Benzo(a)pyrene
- Issue Date
- Environmental Mutagens & Carcinogens (한국환경성돌연변이발암원학회지)
- VOL 21, NO 2, 89-94
- 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.
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