Hoechst 33342 Assay for Nuclear Toxicity

The potential toxicity of the samples (5FU, PBT, and PBT-5FU) at the nuclear level was evaluated by using the Hoechst 33342 staining assay protocol according to the manufacturer’s (Thermo Fisher Scientific, Inc., Waltham, MA, USA) recommendations and to our previous research [14 (link)]. In brief, the malignant cells were seeded in 12-well plates (100,000 cells/1.5 mL/well) and treated with test samples in solvent (media) for 24 h. After the stimulation period, the media was removed, and the staining solution diluted at 1:2000 in PBS was added (500 µL/well). The plates were incubated for 10 min at room temperature, protected from light. Finally, the staining solution was washed with PBS, and the pictures were taken using Cytation 1 (BioTek Instruments Inc., Winooski, VT, USA) and analyzed by the means of the Gen5™ Microplate Data Collection and Analysis Software (BioTek Instruments Inc., Winooski, VT, USA). Staurosporine (STP) 5 µM was selected as a positive control for apoptosis.