Annexin 5 fluorescein 5 isothiocyanate apoptosis detection kit

For apoptosis assays, flow cytometry was performed with an Annexin V-fluorescein-5-isothiocyanate apoptosis detection kit (BD Biosciences, San Jose, CA, USA), according to the manufacturer's protocol. At 48 h after transfection with miR-525 mimics (50 nM) or negative control (50 nM), SW1353 cells were harvested in a 5 ml tube. Then, the cells were washed with cold PBS and resuspended at a final concentration of 1×10⁶ cells/ml. FITC-AnnexinV (5 µl) and propidium iodide were gently mixed and incubated with the cells for 15 min at room temperature. Following incubation, the samples were analyzed using a flow cytoemter within 1 h. Annexin V- and PI+ staining indicated necrotic cells; Annexin V+ and PI+ staining indicated late apoptotic cells; Annexin V+ and PI- staining indicated early apoptotic cells; and the Annexin V- and PI- staining indicated normal cells. Cell apoptosis were determined by flow cytometry using a BD FACSCalibur system (SKU#: 8044-30-1001, BD Biosciences, Franklin Lakes, NJ, USA) and data was analyzed using the ModFit software version 4.1 (Verity Software House, Inc., Topsham, ME, USA).