Parental and transfected cells in the log phase of growth were harvested, centrifuged, and resuspended in binding buffer contains annexin-Vfluorescein isothiocyanate (FITC) (KeyGEN BioTECH, Nanjing, P.R. China) and PI (KeyGEN BioTECH).
Flow cytometric assays were performed using a FACScan flow cytometer (Becton Dickinson). The experiments were repeated three times for each group of cells, and the data were analyzed by FlowJo software (FlowJo, USA).
The apoptotic cell death for the tumor specimens from the in vivo study was examined by the TUNEL method using an in situ cell death kit (KeyGEN BioTECH). The tumor tissue slices were incubated with Proteinase K for 15 min at room temperature, then washed with PBS. Endogenous peroxidase was blocked by 3% H2O2 in methanol for 30 min at room temperature. Sections were then incubated with TUNEL reaction mixture for 60 min at 37°C in a humidified atmosphere in the dark. Positive cells were visualized under fluorescence microscopy. Nuclei were counterstained with a DAPI karyotyping kit (Genmed, Shanghai, P.R. China) and visualized using an FV-1200 laser scanning confocal biological microscope (Olympus, Tokyo, Japan).
Flow cytometric assays were performed using a FACScan flow cytometer (Becton Dickinson). The experiments were repeated three times for each group of cells, and the data were analyzed by FlowJo software (FlowJo, USA).
The apoptotic cell death for the tumor specimens from the in vivo study was examined by the TUNEL method using an in situ cell death kit (KeyGEN BioTECH). The tumor tissue slices were incubated with Proteinase K for 15 min at room temperature, then washed with PBS. Endogenous peroxidase was blocked by 3% H2O2 in methanol for 30 min at room temperature. Sections were then incubated with TUNEL reaction mixture for 60 min at 37°C in a humidified atmosphere in the dark. Positive cells were visualized under fluorescence microscopy. Nuclei were counterstained with a DAPI karyotyping kit (Genmed, Shanghai, P.R. China) and visualized using an FV-1200 laser scanning confocal biological microscope (Olympus, Tokyo, Japan).