Cell cycle and apoptosis analysis

Cell cycle and apoptosis were analyzed by flow cytometry. Briefly, following incubation with DHA (20 µg/ml) or CBP (25 µg/ml) for 24 h, 1×10⁶ LLC cells were harvested with trypsin and washed twice with ice-cold PBS. The cells were fixed with 70% ethanol at 4°C overnight, then treated with propidium iodide and RNase A at 37°C for 40 min. The samples were then analyzed by flow cytometry (FACScalibur; BD Biosciences, San Jose, CA, USA) for the proportion of cells in G₀/G₁, S or G₂/M cell cycle phase. The sub-G₁ population was scored from hypodiploid DNA content. Data were analyzed using ModFit LT V3.2 software (Verity Software House, Topsham, ME, USA). Apoptosis was determined using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit (BioVision, Inc., Milpitas, CA, USA), according to the manufacturer's instructions. The early apoptotic (Annexin V-FITC-positive) and necrotic/late apoptotic (Annexin V-FITC-positive, PI-positive) cells were quantified as apoptotic cells.