2.3. Apoptosis Assay

HUVECs were pretreated with PD (0, 25, 50, and 100 μM) for 2 h, followed by stimulation with MGO (200 μM). In some experiments, HUVECs were pretreated with NAC (10 mM), CsA (1 μM), LY (50 μM), or vehicle control for 2 h before MGO (200 μM) treatment. After 24 h incubation at 37°C in a humidified chamber with 5% CO₂, cells were trypsinized, resuspended in calcium-enriched buffer, stained with Annexin V-FITC and PI for 15 min, and then analyzed by flow cytometry (Guava easyCyte 8HT, Millipore, Boston, MA, USA). Data were calculated with the cell Quest Software. Cell apoptosis was also determined with a TUNEL kit according to the manufacturer's instructions, and all of the nuclei were stained blue with 4′,6-diamino-2-phenylindole (DAPI). The numbers of TUNEL-positive HUVECs and total cells were counted, and apoptosis was evaluated by the ratio of positively stained cells to the total number of HUVECs.