Diversity of peripheral blood mononuclear cells as revealed by a novel multiple microgel "comet assay".

Abstract

Multiple microgel comet assay (MMCA) is a metho-dological adaptation of the single-cell gel electrophoresis assay in which we have introduced the use of standard agarose plug molds in an attempt to improve and expand the applications of the assay. We focused on the study of the heterogeneity of peripheral blood mononuclear cells (PBMC) at the level of the basal single-strand breakage and the DNA damage induction caused by ionizing radiation. Differences among subpopulations were also investigated at the level of chromatin organization and methylation after NotI digestion of microgel-embedded cells. In parallel experiments, the NotI-digested nucleoids were also analyzed with the use of pulsed-field gel electrophoresis (PFGE) and the DNA migration patterns were compared with the corresponding patterns from the MMCA. Significant heterogeneity in the distribution of the oxidative DNA damage, as well as intracellular variations in the NotI digestion patterns were observed in the cell population of PBMC. The combined use of both the comet assay and PFGE provides a useful model for analysis of variation in DNA damage in individual cells as well as information on size of DNA fragments.