2.10. Immunofluorescence staining of γ-H2AX

The subcellular localization of γ-H2AX, the DNA double-strand break marker, was examined in cells (1 × 10⁶ mL^–1) grown on coverslips pre-placed in 6-well plates. Immunofluorescence staining and γ-H2AX nuclear focus quantification were performed as previously described.7 Coverslips were examined under a fluorescent microscope using a green light filter at 450–490 nm for FITC and a blue light filter at 510–560 nm for DAPI. The results were then imported into Image J analysis software to merge the DAPI (blue) and γ-H2AX (green) images. γ-H2AX foci were counted in at least 100 cells for each treatment condition. Cells were considered to be positive (containing prometryn-induced γ-H2AX foci) when more than five green foci were detected.