Canto 2 plus

PC3 cells were seeded at a density of 1 × 10⁵ cells/well and allowed to grow for 24 h. Next, DMSO or AO/854 was added for an additional 48 h. Cells were then harvested and apoptosis was measured using an Annexin V/FITC Apoptosis Detection Kit (KeyGen Biotech, Jiangsu, China). The collected cells were mixed with 5 μL of FITC-Annexin V and incubated for 15 min at 25 °C in the dark. Next, 5 μL of propidium iodide (PI; BD Biosciences, San Jose, Cal., USA) was added and incubated for 5 min in the dark. Three independent replicates were analyzed by flow cytometer (Canto II Plus, BD Biosciences). For cell-cycle analysis, cells were digested with trypsin, collected, and fixed by adding 70% ethanol at 4 °C for 24 h. The cells were stained with PI for 15 min in the dark at 25 °C. The cell cycle was detected by a flow cytometer. All of the results were analyzed by FlowJoV10.

PC3 seeded at a density of 1 × 10⁵ cells/well and developed for 24 h. Subsequently, ML216 or CDDP was added for breeding for an additional 48 h. The Annexin V/FITC Apoptosis Detection Kit (KeyGen Biotech, Jiangsu, China) was used to evaluate apoptosis after the cells were collected. The collected cells were mixed with 5 μL of FITC-Annexin V and incubated for 15 min at 25 °C in the dark. Then 5 μL of propidium iodide (PI; BD Biosciences, San Jose, CA, USA) was added and incubated for 5 min in the dark. Three independent replicates were analyzed using a flow cytometer (Canto II Plus, BD Biosciences). Cells were digested with trypsin, collected, and fixed by adding 70% ethanol at 4 °C for 24 h to conduct a cell cycle study. The cells were stained with PI for 15 min at 25 °C in the dark. A flow cytometer was used to identify the cell cycle. FlowJoV10 conducted a thorough analysis of each outcome.