Annexin-V Apoptosis Assay in A549 Cells

Annexin stain assay was conducted as previously described [19 (link)], with slight modification. Phosphatidylserine (PS) translocation was determined using annexin-V-Fluos apoptosis detection kit (Roche). To each 100 μl A549 cell suspension, 100 μl of Annexin staining buffer and 100 μl of Annexin-V-Fluos labelling solution (annexin-V: propidium iodide: annexin staining buffer (1:1:50 vol/vol/vol)) were added in 1.5 ml tubes. The BD Accuri flow cytometer was used to capture data from stained cells. Cells were analysed using the Accuri C6 gating and CFlow Plus v1.0 analysis software. For each sample 50,000 events were analysed in triplicate. The results are presented as percentage of apoptosis where cells positive for Annexin-V (FITC) in the lower right quadrant (early apoptosis) and upper right quadrant (late apoptosis) were gated.