Apoptosis and necrosis analysis by flow cytometry

Cells were seeded in an appropriate concentration to reach a confluence of 50% up to 80% in 24 h up to 72 h within their cell culture flasks. After 24 h, the incubation medium was changed to a serum-reduced cell culture medium (2% FBS) with different drug concentrations. Concerning talazoparib, cells were treated with an amount of none, 10 nmol/l, 50 nmol/l and 100 nmol/l of inhibitor in each cell culture flask. For niraparib, cells were treated with the amount of none, 1750 nmol/l, 2500 nmol/l and 4000 nmol/l of inhibitor in each cell culture flask. Reduced cell culture medium was used to not artificially enhance the effect of inhibitor. After 3 h, half of the cell culture flasks were irradiated with 2 Gy ionizing radiation (IR) by ISOVOLT Titan X-ray generator (GE, Ahrensburg, Germany). After another 48 h of incubation at 37 °C, cells including the supernatant were harvested. Cells were stained with Annexin V-APC (BD, Heidelberg, Germany) and 7-amino-actinomycin D (7-AAD) (BD, Heidelberg, Germany) for 30 min on ice to analyze apoptosis and necrosis via the Cytoflex flow cytometer (Cytoflex, Beckman Coulter, Brea, USA) [23]. Kaluza Analysis software (Beckman Coulter, Krefeld, Germany) was applied for data evaluation. Cells showing low staining (Annexin-neg./7-AAD-neg.) were defined as “viable” cells, whereas Annexin-pos./7-AAD-neg. Cell were defined as “apoptotic” and double positive cells (Annexin-pos./7-AAD-pos.) as “necrotic” populations (e.g. in Fig. ​Fig.1c,1c, d).