2.3. Apoptotic Assay by Flow Cytometry

Apoptosis was assessed by flow cytometry using externalization of FITC-labeled Annexin V and PI (propidium iodide). Briefly, after treatment with variable concentrations of TF alone or combined with indicated dose of gefitinib for 24 h, human gefitinib resistant NSCLC cell line PC-9R was collected and washed with ice-cold 1 × PBS before staining in 400 μl solution containing Annexin V-FITC for 20 min in dark at room temperature and then staining PI for 5 min. Soon later, the fluorescent signal in the cells was tested by flow cytometry (FACSCanto, Becton Dickinson). Data analysis was showed by the software WinMDI 2.9.