Annexin 5 fitc propidium iodide apoptosis detection kit

The cell apoptosis was tested by Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit (A211-01, Vazyme, China). In brief, AB8/13 cells were plated in a six-well plate and cultured for 48 h. Afterward, the cells were washed with binding buffer and centrifuged at 1,300 rpm for 3 min. Thereafter, the cell pellet was resuspended in 200 μL of binding buffer. Finally, the cells were stained with 5 μL Annexin V-FITC and PI in the dark for 15 min and detected by a CytoFLEX flow cytometer (Beckman Coulter, USA).

The cell proliferation of PCa cell lines was examined by using Cell Count Kit 8 (CCK8) assay as previously described.²² Briefly, the PC3 and DU145 were seeded in 96‐well plates in at least triplicated at a density of 2000 (PC3) or 1500 (DU145) cells/well. The cell proliferation was determined by adding 100 μL of CCK8 reagent (Bimake, Houston, TX, USA) into each well and incubating at 37°C for 2–3 h, and then, the absorbance was measured at 450 nm. For colony formation analysis, 500 DU145 or PC3 cells were seeded in 6‐well plates and cultured for 14 days with routine observation. Then, the cells were washed with ice‐cold PBS, fixed with methanol and stained using crystal violet staining buffer.
For the cell cycle assay, cells were fixed with 75% ethanol at 4°C overnight. The cells were then centrifuged, washed with PBS and stained with propidium iodide (50 μg/mL) in the presence of RNase A for 10 min at 25°C. For cell apoptosis assay, the PC3 and DU145 cells were transfected with siRNAs for 72 h and stained using the Annexin V–FITC/propidium iodide (PI) Apoptosis Detection Kit (Vazyme, China), following the manufacturer's instructions. The cells were analysed using a flow cytometer, and the data were processed by FlowJo V10 (for apoptosis assay) and Modfit (for cell cycle analysis) software.