Single Cell Gel Electrophoresis

The comet assay, also called single cell gel electrophoresis(SCGE), is a sensitive,quick and cheap microscopic method to detect and quantitate DNA damage in eukaryotic cells. It was first described by Ostling and Johanson in 1984 and was later modified by Singh. The comet assay has been used in different fields, including aging, clinical investigations, genetic toxicology and molecular epidemiology in the past few years.We established the procedure of alkaline comet assay in our laboratory. We standardized the protocol of alkaline comet assay step by step, and modified some steps including gel slide preparation, water washing and dehydrationIn order to strengthen the adhesion between thin layers of low melting temperature agarose and a microscope slide, a clean slide need to be dipped in nomal melting temperature agarose for 1 min and then dried at 37 °C over night. Thus a new method of'pouring"gel has been used to substitute the traditional "sandwich"method. In this way, gel's taking off from the slides was avoided.To avoid the the salt and detergent of the lysis solution which interfere the electric field, the slides have to be washed by tridistilled water three times.After the slides dehydrated by grads alcohol, most cells in the gel was located at a similar plane; EB (Ethdium bromide) was selected as the best dye for DNA staining to avoid the fluorescent bleaching. We introduced the CASP software (free) for the quantification image analysis. Thus, an optimized and standard method has been established, the procedure predigested and the time saved.In order to verify the reliability of the method, as well as the feasibility and the reliability of the CASP software,we established a positive model :the DNA damage of K562 cells induced by UV in different times measured by comet assay. A good dose-dependent effect was found, showed the method has a good reliability. The result also suggested that four parameters,Tail Length, Comet Length, Tail Moment and Olive Tail Moment,have a good dose-dependent effect, which showed that the CASP software and the four parameters have a good reliability.Through discussing the primary culture protocols step by step, involving the process of the tissue adhereing to the matrix, the kinds of the medium and the age of animals, we also established the hepatocyte primary culture procedure.Then we tested DNA damage of K562 and hepatocytes induced by magnetic field...