Hoechst 33342

The cells were incubated with CellEvent Caspase‐3/7 Green Detection Reagent (Invitrogen) and Hoechst33342 (Dojindo) for 30 min. Cells were analysed using the In Cell Analyser 2000 with 4′,6‐diamidino‐2‐phenylindole (DAPI) and FITC filters. The ratio of caspase‐3/7‐positive cells to Hoechst33342‐positive cells was determined using the In Cell Analyser Workstation 3.7 software (GE Healthcare).

MDA-MB231 cells were seeded in a 384-well plate (3000 cells/well) for 20 h before the treatment of compounds. After seeding, the tested compounds were diluted in complete media and incubated with cells for 24 h, followed by cytosol and nuclear staining for 1 h with CellTracker Green CMFDA and Hoechst 33342 (Invitrogen), respectively. For cellular tubulin staining, tubulin tracker green was used according to the manufacturer’s instructions (Invitrogen). Cellular images were recorded with an IN Cell Analyzer 6000 (GE Healthcare). After obtaining the images, the nuclear locations and cellular areas were stained with Hoechst 33342 and CMFDA, respectively, and quantitative signals from the images were calculated using a custom-made image analysis algorithm with IN Cell Developer Toolbox (GE Healthcare).

Cell viability and proliferation were detected by Hoechst 33342/propidium iodide (PI) staining using the IN Cell Analyzer 2200 (GE Healthcare, Chicago, IL, USA). Primary mixed glial cells from control and irradiated brains were seeded onto 96-well plates at 10⁴ cells per well and allowed to attach and grow for 24 h. Cells’ viability and proliferation were analyzed at 24, 48, 72, 144, 168 and 192 h after plating. Cells were stained with Hoechst 33,342 (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C and PI (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at 37 °C. An IN Cell Analyzer 2200 cell imaging system (GE Healthcare, Chicago, IL, USA) was used to perform automatic imaging of six fields per well under 200× magnification, in brightfield and fluorescence channels. The images produced were used to analyze live and dead cells using the IN Cell Investigator software (GE Healthcare, Chicago, IL, USA).