In vitro comet test
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In vitro comet test, also known as single-cell gel electrophoresis (SCEG) test, is a sensitive method for rapid detection of DNA damage in single cells, including double-strand breaks, single-strand breaks, and alkali labile sites. The comet assay detects the migration of DNA in an agarose gel following electrophoresis, which results in a “head” of intact DNA and a "tail" of fragmented DNA. The percentage of DNA in the tail of the comet is used as a measure of DNA damage.
In vitro comet assay Procedure
The Human cells are treated with test compounds, and appropriate controls are run alongside the test compounds in the assay. 5 concentrations of the test compound will be applied to the cell for 4hr. The cells are then detached by trypsinization, and embedded in a thin, low-melting-point agarose suspension on a comet assay slide; Cells are further lysed in an alkaline pH buffer to remove cellular proteins and allow for DNA unwinding; The lysed cells are run on gel electrophoresis to separate nucleic acid by size and charge. The broken DNA fragments or damaged DNA migrate away from the nucleus and give out a comet-like appearance after staining with a DNA-specific fluorescent dye with the head being the intact DNA and the tail being damaged DNA fragments. Stained slides are observed under the fluorescence microscopy. The length and the intensity of the comet tail relative to the head reflects the extent and amount of DNA damage. Up to 50 comets per concentration will be analyzed by imaging software to assess the genotoxicity of test compounds.
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