Facscan ow cytometer

The PI-FACS assay was performed to evaluate cell cycle distribution with PI-FACS Cell cycle Detection Kit (MilliporeSigma). In brief, cells were trypsinized, fixed in 70% ethanol, washed once with PBS, and then labeled with propidium iodide in the presence of RNase A for 30 minutes in the dark. Samples were run on a FACScan flow cytometer (Beckman Coulter), and the percentages of cells within each phase of the cell cycle were analyzed using FlowJo software. Three independent experiments were performed.

The Annexin V-APC assay was performed to evaluate cell apoptosis with Annexin V-APC Apoptosis Detection Kit (Invitrogen). Briefly, cells were harvested, washed twice with cold PBS, centrifuged at 189g for 5 minutes and washed with D-Hanks (pH 7.2–7.4). Binding buffer (1×) was used to resuspend cells, and Annexin V-APC was added to the cells and incubated for 15 minutes at room temperature in the dark. Samples were run on a FACScan flow cytometer (Beckman Coulter), and the percentages of apoptotic cells were analyzed using FlowJo software (Beckman Coulter). TUNEL staining of apoptotic cells was detected in glioma cells using a One Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology) following the manufacturer’s protocol. In brief, cells fixed on coverslips with 4% paraformaldehyde for 30 minutes at room temperature and then were treated with 0.1% Triton X-100 for 5 minutes at room temperature. The cells were washed with PBS twice and incubated with 50 μL TUNEL reaction mixture at 37°C for 1 hour. The TUNEL staining cell images were captured using a fluorescence microscope (Zeiss LSM800 confocal microscope). Three independent experiments were performed.