DNA damage

DETEction of DNA damage

Phosphorylation of histone H2AX is a sensitive reporter of DNA damage, particularly DNA double-strand breaks (DSBs). The presence of phosphorylated H2AX (γH2AX) in the nucleus can be detected immunocytochemically. The extent of DNA damage in individual cells may be analysed by flow cytometry on the basis of γH2AX immunofluorescence. It is possible to correlate γH2AX phosphorylation with the position in the cell cycle and/or apoptosis initiation. Such an approach provides a more complete picture of cellular processes upon drug treatment or UV-irradiation.

Flow cytometric detection of cleaved PARP [poly (ADP-ribose) polymerase-1] can also be used as an indication of DNA damage. PARP is an enzyme involved in DNA repair, which is cleaved by active caspase-3 during apoptosis. PARP staining can be achieved via tagging the protein with a fluorescently labelled antibody after UV-irradiation or drug treatment.

To stain for γH2AX or PARP, one can follow the recommendations for phosphospecific flow cytometry posted in the Signaling pathways section.

Another method for DNA damage detection is DNA strand break labelling according to the TUNEL assay. Terminal deoxynucleotidyl mediated dUTP Nick End Labelling is performed in paraformaldehyde fixed cells to keep the DNA in place. Then, to visualise labelled dUTPs (Biotin, BrdU) added by TdT, secondary antibody with fluorochrome is used. Don't forget to counterstain DNA with propidium iodide or other DNA dye.