U937 Cell Apoptosis Assay

Six well plates were used to seed U937 cells at a density of 1 × 10⁵ cells/well for 4 h before treatment with the following concentrations of UD extract: 0.4, 0.8, 1.6, and 2.4%. Untreated cells were used as controls. Cells were collected and centrifuged after 48 and 72 h of treatment, and the pellets were stained with both annexin V and PI (annexin V–fluorescein isothiocyanate (FITC) Apoptosis Detection Kit, Abcam, Cambridge, UK) and immediately analyzed using the Accuri C6 flow cytometer, as previously described [26 (link),27 (link)]. Upon apoptosis induction, phosphatidyl serine is translocated to the outer leaflet of the cell membrane, and living cells exclude PI from the cytoplasm. Therefore, living cells will stain negative for both FITC–annexin V and PI, whereas those that stain positive for both can be classified as apoptotic dead cells [28 (link)].