Since, both A549 and H1793 cell lines provided similar results in response to VJ with respect to inhibition of cell proliferation, therefore, we selected A549 cell line for subsequent analysis. Apoptosis assays were conducted to determine if VJ induced apoptosis in lung cancer cells. For this purpose A549 cells were plated into T-75 flasks and incubated at 37°C for 24 h. Cells were treated with VJ at concentrations 0, 5, 10, 20 or 50 nM for 24 h and were harvested by centrifugation at 1,500 rpm for 5 min and re-suspended in Annexin V binding buffer from FITC conjugated Annexin V Apoptosis Detection Kit (BD Pharmingen) according to supplier’s instructions. To ensure a single cell suspension and to avoid clumps, cells were passed through a 40 µm nylon mesh and diluted to a final concentration of 107 cells/ml. Each sample was analysed using 100 μL of cell suspension. For each concentration of VJ, an independent control was used. To each sample except controls, 2 μL of Annexin V was added, and incubated for 15 min at room temperature in dark. After completion of incubation, 400 μL of binding buffer was added and immediately analysed by FACS analysis using the FACSCalibur (BD Biosciences). The stained and unstained cells were plotted and analysed using FlowJo software as described previously [20 (link)].