MODIFIED NEUTRAL COMET ASSAY FOR HUMAN LYMPHOCYTES

Maria Wojewódzka, Iwona Grądzka, Iwona Buraczewska

Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, 16 Dorodna Str., 03-195 Warsaw, Poland


Comet assay under neutral conditions allows the detection of DNA double-strand breakes, considered to be the biologically relevant radiation-induced lesion. In this report we describe modifications of the neutral comet method, which simplify and facilitate its use for estimation of DNA double strand breaks in human lymphocytes irradiated with doses of 60Co gamma-rays (from 10 to 100 Gy). The analysis carried out according to this protocol takes less time than those published so far. Also, the use of lysis at 50°C is avoided; this is important in view of the presence of heat-labile sites in the DNA of irradiated cells, recently reported by Rydberg. The comets have well defined, sharp limits, are suitable for computer image analysis and chromatin of the control cells remains condensed.