Abstract
The ability of humans to repair DNA damages decreases with increasing age. In order to be able to repair daily occurring DNA damages, it becomes more and more important to preserve repair capability of cells with aging. The preservation of DNA repair processes contributes to preventing DNA mutations and subsequently the onset of age-related diseases such as cancer. For the determination of DNA repair of human cells, mostly in vitro cell cultures are used. However, an ex vivo approach can provide a more accurate result compared with in vitro cell cultures, since the DNA repair ability is measured directly without the influence of prolonged culture time. Published protocols use in vitro cultured cells with a single reporter plasmid or a luciferase reporter. Our modified host cell reactivation assay enables the measurement of DNA repair capacity (nucleotide excision repair) of ex vivo isolated human peripheral blood mononuclear cells (PBMCs). For this purpose, PBMCs are isolated out of human anticoagulated blood by density gradient centrifugation. Directly after isolation, the PBMCs are co-transfected with two plasmids, one being previously damaged by UVC irradiation and one remaining undamaged. PBMCs are incubated for 24 h and subsequently analyzed by fluorescence activated cell sorting (FACS). The ability of cells to repair the DNA damages leads to a functional reactivation of the reporter gene. The assay presented here provides a solution to determine human DNA repair capacity ex vivo directly out of the human body. Furthermore, it can be used to research the ex vivo influence of different substances on DNA repair capacity of humans.