Annexin 5 fitc apoptosis detection assay kit 1

The percentage of apoptotic cells was analyzed by flow cytometry using Annexin V-FITC Apoptosis Detection assay kit I (BD Pharmingen, Lot 25015). Cells (1×10⁵/ml) were washed with ice-cold PBS and resuspended in cold Annexin V-binding buffer containing Annexin V-FITC and propidium iodide (PI). The samples were incubated at room temperature in the dark for 10 min and the total volume was adjusted to 500 μl with Annexin V-binding buffer. The number of stained cells was assessed by flow cytometer (BD FACScan). Early apoptotic cells were defined as positive for Annexin V-FITC but negative for PI staining and late apoptotic cells were positive for both Annexin V-FITC and PI staining.
Cell cycle analysis was performed by fixing cells in 70% ethanol for 12 h at 4°C, followed by incubation with 1 mg/ml RNase A for 30 min at 37°C. Subsequently, cells were stained with PI (50 μg/ml) (Becton-Dickinson, San Jose, CA, USA) in phosphate-buffered saline (PBS), 0.5% Tween-20, and analyzed using a flow cytometer (BD FACScan).

Apoptosis assays were performed by flow cytometry using Annexin V-FITC Apoptosis Detection assay kit I (BD Pharmingen, Lot 25015). Cells were washed with cold PBS and resuspended in cold binding buffer containing FITC-conjugated Annexin V and PI. The mixture was incubated at room temperature in the dark for 15 min and was adjusted to the total volume of 500 μL with Annexin V-binding buffer. The number of stained cells was immediately detected and analyzed using a flow cytometer (BD FACScan). The percentage of early and late apoptotic cells in each group was defined as positive for Annexin V-FITC but negative for PI staining and positive for both Annexin V-FITC and PI staining, respectively.