Annexin 5 fitc staining kit

Cells were harvested using 0.25% trypsin and then washed with 0.1 M PBS. After centrifugation at 800 × g for 5 min, cells were treated with 100 μl binding buffer and then stained with 2 μl propidium iodide and 2 μl annexin V-fluorescein isothiocyanate for 15 min. In this study, an annexin-V-FITC staining kit (Roche Applied Science) was used to assess cell apoptosis, we used the BD FACS Calibur flow cytometry (BD Bioscience, San Jose, CA) and the MACSQuant flow cytometry (Miltenyi Biotechnology, Bergisch Gladbach, Germany) to examine the cell apoptosis. Three independent flow cytometric experiments were performed.

Cell killing assayed was performed as described previously with a modification [15] (link), [26] (link)–[28] (link). The PI-exclusion method for loss of integrity of cell membranes was used to assess viability. In brief, cells were suspended in PBS containing 25 µg/ml PI for 5 min at 37°C and then subjected to analytic flow cytometry on a FACSort (BD Biosciences) immediately after labeling. A light scatter gate was set up to eliminate cell debris from the analysis. The PI fluorescence signal was recorded on the FL3 channel and analyzed by using CellQuest software. Phosphatidylserine (PS) translocation, which precedes loss of PI exclusion in apoptotic cell death, was assessed by an annexin V-FITC staining kit (Roche) following the manufacturer’s protocol. For nuclear morphology, cells were stained with DAPI and apoptotic cell (nuclei condensation) were visualized under UV microscope.