Annexin 5 fitc apoptosis analysis kit

Cells (2 × 10⁵ cells/well) were seeded into 6-well plates and cultured for 24 h. The compounds (NC, GSK343, DZNep, gefitinib, G + g, and D + g) were added at various indicated concentrations for 48 h. For the cell apoptosis assay, cells were stained using the Annexin V-FITC Apoptosis Analysis Kit (BD Biosciences, San Jose, CA, USA) and were analyzed using the FACSAria™ flow cytometer (BD Biosciences). For the cell cycle assay, cells were trypsinized and fixed with 70% ice-cold ethanol overnight. Subsequently, cells were treated with DNase-free ribonuclease (TaKaRa, Beijing, China), stained with propidium iodide (PI; BD Biosciences), and analyzed using the FACSAria™ flow cytometer (BD Biosciences) equipped with ModFit LT (Topsham, ME, USA).

Cells (2 × 105 cells/well) were seeded into 6‐well plates and cultured for 12 h. Cisplatin and/or PD‐0332991 were added at various concentrations and cells were cultured for another 24 h. Cells were stained using an Annexin V‐FITC Apoptosis Analysis Kit (BD Bioscience) and analyzed with a FACSAriaTM flow cytometer (BD Bioscience).

For analysis of DNA contents, cells were trypsinized, fixed in 80 % ethanol for 16 hours, and treated with RNaseA (100 μg/ml) at 37 °C for 2 hours. Cells stained with propidium iodide (40 μg/ml) were analyzed by flow cytometry with 30,000 events (FACScaliber, Becton Dickinson). For analysis of cell death, we followed the manufacture's manual of Annexin V-FITC apoptosis analysis kit (BD Pharmingen). Briefly, cells were trypsinized and washed by ice-cold PBS twice. 1 × 10⁵ cells are suspended in 100 μl of Binding buffer, and 5 μl of Annexin V-FITC (BD Pharmingen) and propidium iodide were added. After incubation for 15 min, 400 μl of Binding buffer were added, and analyses were carried out using a flow cytometry. Data were processed with WinMDI software.

Apoptosis of the HD11 cells exposed to Fe₃O₄-NPs of various sizes and concentrations was measured using an Annexin V-FITC apoptosis analysis kit (BD, NJ, USA) according to the manufacturer's instructions. HD11 cells in 12-well plates were incubated with Fe₃O₄-NPs of different sizes at different concentrations in 1640 medium with 10% FBS. After 24 h of exposure, the cells were collected and washed twice with cold PBS while being stirred at 1000 r.p.m. for 5 min. Then, the cells were adjusted to a concentration of 1 × 10⁶ cells ml⁻¹ with 1 × binding buffer. A total of 100 µl of cell suspension was placed in a 5 ml Falcon tube, and 5 µl of FITC Annexin V was added to each tube in the dark for 20 min at room temperature. Then, 5 µl of PI was added to each tube in the dark for 5 min, and 400 µl of 1 × binding buffer was added to each tube. All samples were analysed using flow cytometry (BD LSRFortessa™, USA) within 1 h.