Analysis of cell apoptosis by TUNEL assay

MCF-7 cell apoptosis was determined with an In Situ Cell Death Detection Kit (Roche, Merck KGaA, Darmstadt, Germany). According to the manufacturer’s protocol, cells were fixed with 4% paraformaldehyde for 1 hour. Terminal deoxynucleotidyl transferase solution was dissolved in the dark, added to the cells, and incubated for 60 minutes at 37°C. Then, the cells were washed twice in PBS buffer for 5 minutes each time. TUNEL-positive cells (green) that were also dyed with DAPI (blue) were identified as apoptotic cells under a fluorescence microscope. The percentage of apoptotic cells was determined by counting the TUNEL-positive cells and dividing the number by the total number of cells.