Detection of apoptotic cells

Cells were grown to 40–50% confluence in 6-well plates and drugs were added next day. After 24 h (Cisplatin, Camptothecin, Doxorubicin) or 48 h (Etoposide) cells were trypsinized, span down and washed twice with PBS. Washed cells were suspended in 100 μl of Annexin V Binding Buffer (BD Pharmingen) containing 5 μl of Annexin V conjugated with Alexa Fluor^®647 fluorophore (Biolegend) and 5 μl of 7-Aminoactinomycin D (7-AAD, BD Pharmingen) or GelGreen dye (Biotium) diluted 1:10⁴. After 20 min 400 μl of Annexin V Binding Buffer was added and probes were analysed with flow cytometer (BD FACSCalibur, BD Biosciences) using FL1 (GelGreen dye), FL3 (7-AAD) or FL4 (Alexa Fluor^® 647 Annexin V) detectors. Data analysis was carried out with FCS Express 4 Software.