Cell apoptosis analysis

A549 or SPC-A-1 cell suspensions (5×10⁵ cells/ml) and Annexin V-FITC (Abcam, Shanghai, China) binding buffer was prepared. An Annexin V-FITC Apoptosis Detection kit (cat. no. APOAF-20TST; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used according the manufacturer's protocol. Annexin V-fluorescein isothiocyanate (FITC; 0.5 mg/ml) was added to the cell mixture and incubated at room temperature for 15 min. Subsequently, propidium iodide (PI; 0.1 µg/ml; Abcam) was added for 5 min at room temperature, and samples were analyzed on a fluorescence-activated cell sorting analyzer instrument using the 488 nm excitation wavelength (argon-ion laser or solid state laser) and emission detected was at 530 nm (green, FITC) and 575–610 nm (orange, PI) (28). Data were analyzed with BD FACSuite™ software (23–12943; BD Biosciences, San Jose, CA, USA).