TUNEL Assay

TUNEL assay (TdT-mediated dUTP Nick-End Labeling) (Roche) was used to label fragmented DNA in cells. Fluorescein-12-dUTP binds to the 3’-OH of the DNA strand and detected by a fluorescence microscope. Cells were cultured in glass coverslips and fixed in 3% PFA in PBS for 25 min at RT. PFA was removed, and 200 mM glycine was added for 15 min. Then, cells were permeabilized with 0.2% triton X-100 for 20 min. After that, cells were blocked with 1% BSA in PBS with 0.1% sodium azide for 1 h at RT, or overnight at 4°C. 50 μl of TUNEL reaction mixture was added (prepared according to the manufacturer). Coverslips were incubated for 1 h at 37°C in darkness. Coverslips were washed 3 times with PBS. Nuclei were stained with DAPI (4’, 6-diamidino-2-phenylindole), which was added at 1:1,000 dilution for 15 min in the dark. Finally, coverslips were washed 3 times with PBS, and mounted on microscope slides with MOWIOL 4-88 (Calbiochem, MA, United States). Samples were visualized using a Leica TCS SP5 inverted fluorescence confocal microscope and analyzed by ImageJ software.