Alaxa fluor 488 annexin 5 dead cell apoptosis kit

Human ovarian cancer cell lines A2780/CP70 and OVCAR-3 were obtained from Dr. Jiang at West Virginia University. Normal ovarian surface epithelial cells IOSE-364 were provided by Dr. Auersperg at the University of British Columbia, Canada. All cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, and the cells were grown in a humidified incubator with 5% CO₂ at 37°C. RPMI-1640 medium, Bovine Serum Albumin, DMSO, Hoechst 33342, DCFH-DA and pifithrin-α (PFT-α) and were purchased from Sigma-Aldrich (Sigma, St. Louis, MO, USA). CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay was obtained from Promega (Madison, WI, USA). Pierce LDH Cytotoxicity Assay Kit and Alaxa Fluor® 488 Annexin V/Dead Cell Apoptosis kit were purchased from Thermo Scientific (Waltham, MA, USA). Propidium iodide (PI), phosphate-buffered saline (PBS) and fetal bovine serum (FBS) were obtained Life Technologies (Invitrogen, Grand Island, NY, USA). Primary antibodies against PCNA, Cyclin A, CyclinD1, Cyclin E, Cdc25A, CDK4, γ-H2AX (Ser139), p21 Waf1/Cip1(12D1) were purchased from Cell Signaling Inc. (Danvers, MA, USA). Primary antibodies against pChk2 (Thr68), Chk2 (H-300), p53 (C11), p-p53 (Ser15), GAPDH (0411) and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cells were seeded in 60 mm dish palate at a density of 1×10⁵ cells/well, after a 24 h growth for adhesion, cells were treated with different concentrations of TFS for 24 h and used for further analysis. For apoptosis analysis, an Alaxa Fluor® 488 Annexin V/Dead Cell Apoptosis kit (Thermo Scientific, MA, USA) was used in the experiment. Briefly, the treated cells were harvested and washed twice with cold PBS, followed by resuspending the cells in propidium iodide and annexin V buffer according to the manufacturer’s instruction. Samples were analyzed using a FACSCaliber flow cytometry system (BD Biosciences, USA).