Evaluating Senescence in Cell Treatments

To investigate evolving senescence during KI or irradiation treatment, we seeded cells at low confluence in cell culture flasks. After 24 h of settlement, cells were treated with either kinase inhibitor, irradiation, or combination of both. As a control, cells were treated with DMSO only. On day 6 after treatment, medium was exchanged including DMSO or kinase inhibitor again. After 10 days of treatment, cells were collected and stained as previously published (23 (link)). Briefly, cells were treated with 100 nM Bafilomycin A1 (Merck, Darmstadt, Germany) for 30 min (37°C). Afterwards, Hoechst dye was added for another 30 min (37°C) and finally cells were treated with C12-FDG for 60 min (37°C). After centrifugation cells were resuspended in Ringer solution and stained with Annexin V-APC and 7-AAD (BD, Heidelberg, Germany) for 30 min on ice. Finally, C12-FDG positive cells were measured using flow cytometry (Cytoflex S, Beckman Coulter, Brea, CA, USA).