Opera phenix high content analyzer

Cells (2 × 10⁴ cells, 100 μL/well) were seeded into 96-well plates (Perkin Elmer, Waltham, MA, USA) and grown for 24 h. The following day, cells were pretreated with 100 μM z-DEVD-FMK caspase inhibitor (#S7312, Selleckchem, Houston, TX, USA) and/or treated with 5 µM cisplatin (Accord, Warsaw, Poland). Then, the cells were stained with 50 μL CellEvent™ Caspase-3/7 Green Detection Reagent (Table S2) in 7 μM final concentration. The plates were placed into an Opera Phenix High-Content Analyzer (Perkin Elmer, Waltham, MA, USA) with environment control (5% CO₂, 37 °C) and fluorescence was measured every hour for 11 h at 502/530 nm. The analysis was performed with the Harmony software (Perkin Elmer, Waltham, MA, USA).

Spheroids were transferred to glass bottom microplates (Cell Carrier-96 ultra, PerkinElmer, Waltham, MA, USA) and treated with cisplatin (25, 50, and 100 μg/mL) on day 2 and day 4. Afterward, the cells were stained with Hoechst (Table S2) and Annexin V-FITC (Table S2.) for 1 hour in growth medium. Images were acquired using an Opera Phenix High Content Analyzer (Perkin Elmer, Waltham, MA, USA). Fluorescence intensity was detected at 350 (Hoechst) and 488 nm (Annexin V-FITC).

Cells (2 × 10⁴, 100 μL/well) were seeded into sterile microplates (Cell Carrier-96 ultra, PerkinElmer, Waltham, MA, USA) and grown for 24 h. After applying the indicated cell treatments (see figure legends), cells were fixed in 3% formaldehyde/PBS solution for 15 min at room temperature, washed 3× with PBS, and incubated in blocking solution (5% BSA in PBS) for 15 min at room temperature. The anti-phospho-H2AX antibody (Table S2) was diluted in blocking solution and added to wells (50 μL/well, 2 h at room temperature). The secondary antibody (Alexa Fluor 488, Table S2) was diluted in blocking solution and incubated for 1 hour. After antibody incubations, cells were washed twice with PBS and incubated for 5 min with PBS containing 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI, Table S2) at room temperature for nuclear staining. Cells were then washed three times with PBS and were kept in 100 μL of PBS until imaging. Images were acquired using an Opera Phenix High Content Analyzer (PerkinElmer, Waltham, MA, USA) with a 10× air objective (NA 0.3). Image analysis was performed with the built in Harmony software (version 4.8).