Apoptosis detection by simultaneous acridine orange/ethidium bromide (AO/EtBr) and Hoechst staining

About 10000 cells were seeded in 35 mm Petri dishes (Nunc™ Dishes) and incubated under culture conditions for 24 hours. After treatment with different concentrations of the drugs, the plates were incubated 48 hours and 72 hours for DOX and MTX treatment, respectively.

For cell fixation, 4% paraformaldehyde (PFA)/4% sucrose in phosphate buffer (PBS) was added to the Petri dishes for 15-45 minutes. The cells were then washed with 0.1% triton X-100 (Sigma,USA) in PBS for 5 minutes. First of all, we stained the fixed cells with 2.5 µg/µl AO/EtBr (Sigma, USA) in PBS/0.1% triton X-100 for 5 minutes. Afterwards, the cells were stained with 2.5 µg/µl Hoechst 33342 (Sigma, USA) in PBS/0.1% triton X-100 and checked by fluorescence microscopy (Axoscope 2 plus fluorescence microscopy-ZEISS, software Infinity capture, Germany) (15).