Apoptosis Assay Using Annexin V-FITC and Propidium Iodide (PI) Dual Staining

In order to carry out an apoptosis assay by flow cytometry, MRC-5 and A549 cells were seeded at a density of 1 × 10⁵/ml in 25 cm² flask overnight before being treated LPV/r at various concentrations for 24–48 h. Camptothecin (CPT) (50 μM) (Sigma) treatment was used as a positive control to induce apoptosis. Determination of apoptotic cell numbers by fluorescent staining was done using the Annexin V FITC/PI apoptosis kit from Santa Cruz Biotechnology, following manufacturer's instructions. Briefly, cells were incubated in triplicate with Annexin V FITC and propidium iodide (PI) in binding buffer for 15 min in the dark; and stained cells were immediately subjected to flow cytometry analyses using the BD C6 Accuri flow cytometer (BD Biosciences).