Dual Immunofluorescence of gamma H2AX and 53BP1 in Human Peripheral Lymphocytes

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, including cancer. The cell is, therefore, provided with complex mechanisms to repair DSBs, and these pathways involve histone H2AX in its phosphorylated form at Ser-139 (namely gamma H2AX) and p53 binding protein 1 (53BP1). As both proteins can form foci at the sites of DSBs, identification of these markers is considered a suitable method to study both DSBs and their kinetics of repair. According to the molecular processes that lead to the formation of gamma H2AX and 53BP1 foci, it could be more useful to investigate their co-localization near the DSBs in order to set up an alternative approach that allows quantifying DSBs by the simultaneous detection of two DNA damage markers. Thus, this protocol aims to assess the genomic damage induced in human lymphocytes by the radiomimetic agent bleomycin through the presence of gamma H2AX and 53BP1 foci in a dual immunofluorescence. Using this methodology, we also delineated the variation in the number of gamma H2AX and 53BP1 foci over time, as a preliminary attempt to study the repair kinetics of bleomycin-induced DSBs.

Automated summary

Double strand breaks (DSBs) are severe DNA lesions that can lead to cancer if not repaired. This study focuses on the use of gamma H2AX and 53BP1 foci as markers to study DSBs and their repair kinetics. By detecting the co-localization of these markers near DSBs, a dual immunofluorescence protocol is established to quantify DSBs induced by the radiomimetic agent bleomycin in human lymphocytes. The variation in the number of gamma H2AX and 53BP1 foci over time is also examined to understand the repair kinetics of bleomycin-induced DSBs.