Live dead cell double staining kit

A Live/Dead Cell Double Staining Kit (KGAF001, KeyGEN BioTECH, Jiangsu, China) was used to assess cell viability following the manufacturer’s instructions. First, 5 μl of reagent A (PI) and B (calcium AM) were mixed with 10 ml of PBS. Then, the cells were washed twice with sterile pre-heated PBS, the prepared staining solution described above was added, and the cells were incubated at room temperature for 5–10 min. Finally, the cells were washed twice with PBS and then observed under a fluorescence microscope in time.

PSOs were cultured in 96-well plates, with 2-3 PSOs per well. Eight wells constituted each test group, and each test group was treated with various chemotherapeutic drugs or targeted drugs, such as paclitaxel (A71384, Innochem, China), sirolimus (53123-88-9, MedChemExpress, USA), oxaliplatin (A27295, Innochem, China) and capecitabine (C124969, Aladdin, China). The drugs were administered at the respective clinical-relevant concentrations, as indicated in the figure legends, for a period of 5-7 days. Subsequently, Live/Dead cell double staining kit (KGAF001, KeygenBiotech, China) was used to stain the PSOs, per the manufacturer’s specifications. After staining, the PSOs were visualized using a fluorescence microscope (BDS400, Cnoptec, China) with 500-550nm excitation light. The green and red fluorescence emissions were isolated and analyzed separately. The mean fluorescence intensities of each group were then quantified using ImageJ software (version v1.53h, NIH).