4.4. Analysis of Apoptotic Cell Death Induction

To investigate induction of apoptotic cell death, cells were plated in 6-well plates and after a recovery period of 4 h, cells were treated with volasertib (0–20 nM) for 24 h. Subsequently, cells were irradiated (0–8 Gy) and washed with drug-free medium. Seventy-two hours later, adherent plus floating cell fractions were collected and apoptotic cell death was evaluated using the annexin V-FITC apoptosis detection kit (Becton Dickinson, Franklin Lakes, NJ, USA), according to the manufacturer’s instructions. Flow cytometric analysis was performed on the FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Each sample was analyzed using 10,000 events/sample. Data were analyzed using FlowJo Software v.10.0.7 and presented as dot plots (annexin V plotted against PI staining). For A549-NTC and A549-920 cells, the number of apoptotic cells was determined using annexin V-APC due to the interference of FITC with the control protein turbo-GFP present in the vector.