title: Direct use of the comet assay to study cell cycle distribution and its application to study cell cycle-dependent DNA damage formation.
abstract: The comet assay or single cell gel electrophoresis has proven to be a versatile and sensitive method of measuring the induction and repair of DNA damage in individual cells. However, one of the drawbacks of the assay is the bias caused by changes in the ability of cells to repair DNA damage in different cell cycle phases. Whereas the bias seems less important when G0 peripheral blood lymphocytes are studied, it might cause problems when proliferating cells are investigated. In this paper, we validate the assumption that the total comet fluorescence intensity corresponds to the position of the cell in the cell cycle and can be used to assign single cells to specific cell cycle phases. To validate the approach, we used a very homogenous blood mononuclear CD34(+) cell population in G0 phase (unstimulated) or stimulated to enter the cell cycle. An analysis of the cell cycle distribution revealed that the 15 comet intensity classes and the 100 comets usually analyzed in a typical comet experiment are sufficient to obtain a reliable cell cycle distribution comparable with the results obtained by the flow cytometry for the same cell population. The effect of the cell cycle position on the results obtained by the comet assay for proliferating and non-proliferating cell populations irradiated with 3 Gy of X-radiation is also discussed.
authors.affiliation: Centre for Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, 16 Dorodna Street, 03-195, Warszawa, Poland. m.kruszewski@ichtj.waw.pl
authors.forename: Marcin
authors.initials: M
authors.lastname: Kruszewski
authors.forename: Teresa
authors.initials: T
authors.lastname: Iwaneńko
authors.forename: Eugeniusz K
authors.initials: EK
authors.lastname: Machaj
authors.forename: Tomasz
authors.initials: T
authors.lastname: Ołdak
authors.forename: Maria
authors.initials: M
authors.lastname: Wojewódzka
authors.forename: Lucyna
authors.initials: L
authors.lastname: Kapka-Skrzypczak
authors.forename: Zygmunt
authors.initials: Z
authors.lastname: Pojda
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism; D002453:Cell Cycle / Q000528:radiation effects; D020552:Comet Assay / Q000379:methods*; D004249:DNA Damage / Q000528:radiation effects; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000378:metabolism / Q000528:radiation effects; D006801:Humans; D016130:Immunophenotyping; D007963:Leukocytes, Mononuclear / Q000378:metabolism / Q000528:radiation effects; D014965:X-Rays
chemical_list: D018952:Antigens, CD34