Pi solution

LNCaP and C4-2 cells (1×10⁶) were trypsinized, washed twice with PBS and fixed in 70% ice-cold ethanol (Sangon Biotech Co., Shanghai, China) for 1 h at 4°C. The samples were centrifuged at 300 × g for 5 min at 4°C, the ethanol was removed and they were incubated with 100 mg/ml RNaseA (Sigma-Aldrich; Merck KGaA) for 30 min at 37°C. For cell-cycle distribution analysis, 400 ul PI solution (BestBio Co., Ltd., Shanghai, China) was added to the cells. The cells were vortexed and incubated in the dark for 30–60 min at 2–8°C. For cell apoptosis analysis, cellular DNA was stained with the Annexin V-FITC/PI Apoptosis Detection kit (cat. no. BB-4101-2; BestBio Co., Ltd., Shanghai, China). Briefly, the cells were centrifuged at 300 × g for 5 min at room temperature and resuspended in Annexin V Binding Buffer. A total of 5 µl FITC Annexin V was added to the suspension, they were then gently vortexed and incubated for 15 min at 2–8°C in the dark. The cells were then incubated with 10 µl of PI solution, gently vortexed and incubated for 5 min at 2–8°C in the dark. Cell-cycle distributions and cell apoptosis were determined by flow cytometry using a BD FACSCalibur system (BD Biosciences, Franklin Lakes, NJ, USA) and data was analyzed using the ModFit software version 4.1 (Verity Software House, Inc., Topsham, ME, USA).