2.6. Annexin V/Propidium Iodide (PI) Staining-Based Apoptosis Assay

The Annexin V-FITC Apoptosis Kit was used to quantify cell apoptosis. Cells (1 × 10⁶/well) were seeded in 6-well plates and treated with various concentrations (0, 9, and 15 μM) of SSD for 48 h. The samples were digested with ethylenediaminetetraacetic acid- (EDTA-) free trypsin, washed thrice with phosphate-buffered saline (PBS), thoroughly mixed with a blocking buffer, and stained with Annexin V-FITC conjugate for 10 min and PI for 5 min before fluorescence-activated cell sorting analysis (BD Biosciences, San Jose, CA, USA). Cells were classified as viable (Annexin−/PI−), early apoptotic (Annexin+/PI−), late apoptotic (Annexin+/PI+), and necrotic (Annexin−/PI+).