The PSH-induced apoptosis rate of U251 cells was determined using Annexin V-FITC/PI staining. U251 cells were seeded into 6-well plates at a density of 1×106 cells/well and cultured for 24 h at 37°C. Following the aforementioned treatments, the cells were washed with PBS, trypsinized with 0.125 g/l trypsin, centrifuged at 800 × g for 5 min at room temperature and collected. The cells were resuspended in 100 µl 1X binding buffer, and 5 µl Annexin V-FITC and 5 µl PI were added to each well and incubated for 15 min at 37°C in the dark. Subsequently, 400 µl binding buffer was added, and apoptosis was analyzed on a FACSCalibur flow cytometer with BD Accuri™ C6 software (both BD Biosciences). A total of 1×104 cells were collected per well, and early and late apoptotic cells were recorded.

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