Annexin 5 fluorescein isothiocyanate fitc propidium iodide apoptosis assay kit

Human bladder cancer cell lines T24 and 5637 were provided by the cell bank of the Chinese Academy of Sciences in Shanghai. The present study used metformin (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China), Roswell Park Memorial Institute (RPMI)-1640 medium (Cellmax Company, Groningen, Netherlands), fetal bovine serum (FBS) (Sigma, St. Louis, MO, USA), phosphate-buffered saline (PBS) (Beijing Solarbio Company, Beijing, China), Cell counting Kit-8 (CCK-8) (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China), and an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Assay Kit (BD Biosciences, San Jose, CA, USA). Antibodies recognizing PI3K/Akt/mTOR signaling pathway associated proteins (phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), phosphorylated (p)-PI3K, protein kinase B (Akt), p-Akt, mechanistic target of rapamycin (mTOR), p-mTOR), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Apoptosis-related protein (cleaved-caspase 3 and cleaved-poly(ADP-ribose) polymerase (PARP)) were all purchased from Affinity Biosciences (Cincinnati, OH, USA). We also used a microplate analyzer (Thermo Scientific, Waltham, MA, USA), a flow cytometer (Bio-Rad, Hercules, CA, USA), and a western blotting imaging system (Bio-Rad).

Cell apoptosis was detected with the Annexin V-fluorescein isothiocyanate (FITC)/ propidium iodide (PI) apoptosis assay kit (BD Biosciences). Cell lines T29, A2780 and HEY were incubated with DMSO (diluent), PHPO, cisplatin or PHPO + cisplatin at the concentrations of IC50s for 24 h. The cells were harvested, washed twice with cold 1 × PBS and resuspended in 500μL binding buffer at a density of 1 × 10⁵cells/mL. The cells were then stained with 5μL Annexin V-FITC and 5μL PI for 15min in dark condition at room temperature and subjected to analysis by flow cytometry (Cytomics FC 500MPL, Beckman Coulter). The early apoptosis was evaluated based on the percentage of cells with Annexin V + / PI - (the right lower quadrant, representing early apoptosis), while the late apoptosis was that of cells with Annexin V + / PI + (the right upper quadrant, representing late apoptosis). The results were indicated as mean values from three independent determinations.