Apoptosis Detection by Annexin V-FITC/PI Dual Staining

Annexin V-fluorescein isothiocyanate/propidium iodide dual staining was employed to distinguish early and late apoptotic cells (BD Pharmingen, San Jose, CA, USA). Fluorescein isothiocyanate -conjugated Annexin V was used to quantify the loss of asymmetry of phosphatidylserine on cell membranes involved in apoptosis while propidium iodide differentiates the early apoptotic, the late apoptotic and necrotic cells [22 (link)]. Briefly, HT29 cells (3 × 10⁵ cells/well) were seeded in a 6-well plate and treated with the orientin for 48 h. Cells were gently washed twice with phosphate buffered saline accompanied by tryptic digestion (0.25% trypsin/EDTA) and washed yet again with PBS. All the cells including the floating and adherent ones were harvested, pooled and incubated with dual stain for 15 min on ice under dark. The cells were counted by a flow cytometer (Becton-Dickinson, San Jose, CA, USA).