Annexin V/Apoptosis Analysis Using Flow Cytometry

Annexin V/propidium iodide (PI) (Sigma Chemicals, U.S.) assay was carried out to quantify viable, apoptotic (early and late), and necrotic cells using flow cytometry. H₂O₂ induced oxidative stressed HUVECs (1 × 10⁵/well) were plated in a 24-well plate and incubated with BSE, CSE, and BSE-SLNPs (2 and 4 μg/ml) or vehicle control for 48 h. After incubation, cells were incubated in 400 μl of 5 μl Annexin V-FITC and 5 μl PI containing binding buffer, and then the cells were kept in the dark for 15 min at room temperature (RT). The cells were analyzed by flow cytometry (BD Biosciences, San Jose, CA) to identify the apoptotic (Annexin V positive and PI negative) and late apoptotic (Annexin V positive and PI positive) cells.