Apoptosis assessment by DAPI staining

Harvested EAC cells (1 ml) from each mouse were centrifuged at 1200 rpm for 2 min and collect the plate from supernatant. The plate was then washed with PBS for each time followed by centrifugation at 1200 rpm for 2 min for three times. The resultant cells were incubated with 5 μl DAPI staining solution in the dark for 10 min with subsequent adding of PBS to the DAPI containing pellet and then centrifuged at 1200 rpm for 2 min. Finally, 200 μl PBS was added to the pellet and 10 μl of the supernatant was taken in a microscopic slide and observed the morphological changes of EAC cells with fluorescence microscope.