In the present research, 72 hrs after cell transfection, cells were digested using the trypsin free of ethylenediaminetetraacetic acid (EDTA), and collected and re-suspended at a concentration of 106 cells/mL after three times of washing with 1 mL ice-cold phosphate-buffered saline (PBS). According to the instructions provided for the Annexin V-PE Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA), 5 μL of PE-Annexin V, and 5 μL of 7-AAD were added to each 100 μL of diluted binding buffer. After the cells were incubated at room temperature for 15 mins, cell apoptosis was detected by flow cytometer FC500/FC500-MPL (Beckman Coulter, Brea, CA, USA).
For cell cycle analysis, cells were added into 1 mL pre-cooled 75% alcohol (−20°C), and fixed at temperature of 4°C overnight after cell transfection for 72 hrs or 120 hrs. Cells were re-suspended in 500 μL propidium iodide (PI)/RNase Staining Buffer (BD Biosciences, San Jose, CA, USA) after twice washing with cold PBS. Following 30 mins of incubation in the dark at room temperature, cells were analyzed by flow cytometer FC500/FC500-MPL (Beckman Coulter, Brea, CA, USA).
For cell cycle analysis, cells were added into 1 mL pre-cooled 75% alcohol (−20°C), and fixed at temperature of 4°C overnight after cell transfection for 72 hrs or 120 hrs. Cells were re-suspended in 500 μL propidium iodide (PI)/RNase Staining Buffer (BD Biosciences, San Jose, CA, USA) after twice washing with cold PBS. Following 30 mins of incubation in the dark at room temperature, cells were analyzed by flow cytometer FC500/FC500-MPL (Beckman Coulter, Brea, CA, USA).