Annexin 5 fluorescein isothiocyanate propidium iodide kit

Following transfection, cells were collected and washed with phosphate-buffered saline before re-suspending 1 • 10 6 cells from each sample in binding buffer. Binding buffer contained phosphate buffer saline (PBS) and 1% FBS. Then, an Annexin V-fluorescein isothiocyanate/propidium iodide kit (BD Pharmingen, San Diego, CA) was used to measure cell viability via flow cytometry (BD FACS Aria; BD Biosciences, Franklin Lakes, NJ). After transfection, PTC cell lines (1 • 10 4 ) were re-suspended in 200 lL of serum-free medium. For both invasion and migration assays, PTC cell lines were then seeded into the upper chambers of Transwell Ò plates (8 lm pore size; Corning Costar), which were coated with or without Matrigel (BD Biosciences). Medium containing 10% FBS was used as a chemoattractant and added to the bottom chamber of each well, and cells were incubated at 37°C with 5% CO 2 for 24 or 48 h for migration and invasion assays, respectively. Afterward, cells present in the upper chamber were wiped out with cotton swabs, whereas cells that had adhered to the bottom chamber were fixed with methanol and stained with 0.1% crystal violet. Stained cells were imaged at 100 • magnification under a microscope (Olympus, Tokyo, Japan).