Annexin 5 fluorescein isothiocyanate fitc propidium iodide apoptosis kit

Apoptosis was assessed using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit (BioVision, Inc., Milpitas, CA, USA), according to the manufacturer's protocol. Briefly, 1×10⁵ cells were seeded into 6-well plates and cultured overnight, prior to exposure to various concentrations of angelicin for different times (HepG2 cells 48 h and Huh-7 36 h). HepG2 cells were treated with 200 nM LY294002 in the absence or presence of angelicin for 24 h. Subsequently, cells were collected and incubated with Annexin V-FITC/PI, according to the manufacturer's protocol. Apoptosis was assessed using a Guava^® easyCyte Flow Cytometer (EMD Millipore, Billerica, MA, USA), and the data were analyzed using CellQuest Pro software (version 5.1, BD Biosciences, Franklin Lakes, NJ, USA).

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.