Annexin 5 fitc detection kit

An apoptosis assay was performed using the Annexin V–FITC Detection Kit (Roche, Switzerland) according to the manufacturer’s instruction by flow cytometry. For the cell cycle assay, HT-29 and HCT116 cells were cultured in 6-well plates at 2.5 × 10⁵ cells/well, and harvested after 24 and 48 h of exposure to bigelovin at indicated doses (0.7–5.4 μM). Cells were washed with PBS and ice-cold 70% v/v ethanol was used to permeabilize cell membrane at 4 °C overnight. After permeabilization, cells were resuspended in PBS containing RNase A (10 μg/ml) and PI (20 μg/ml) for 30 min in the dark at 37 °C and then analyzed by flow cytometry (10,000 events).

For cell cycle analysis, the indicated cells were synchronized by serum starvation for 12 h and induced to reenter the cell cycle by an exchange of 10% FBS. Cells were then harvested at 48h after replacing the medium and fixed in ice-cold 70% ethanol at 4°C overnight. Subsequently, cells were stained with propidium iodide solution (50 μg/mL propidium iodide, 50 μg/mL RNase A, 0.1% Triton-X, 0.1 mM EDTA), and subjected to FACS analysis (BD Biosciences, NJ).
For apoptosis analysis, the indicated cells were harvested, washed with PBS, suspended in binding buffer, and sequentially stained with Annexin V-FITC Detection Kit (Roche Applied Science, Penzberg, Germany) by flow cytometry. Each experiment was performed in triplicate.

For cell cycle analysis, 2 day after siRNA transfection, cells were synchronized by serum starvation for 12 h and induced to reenter the cell cycle by an exchange of 10% FBS. Following this, cells were harvested at 24, 48, and 72 h after replacing the medium and fixed in ice-cold 70% ethanol at 4°C overnight. Cells were then stained with propidium iodide solution (50 μg/mL propidium iodide, 50 μg/mL RNase A,0.1% Triton-X, 0.1 mM EDTA), and subjected to FACS analysis (BD Biosciences, NJ).
For apoptosis analysis, the indicated cells were harvested, washed with PBS, suspended in binding buffer, and sequentially stained with Annexin V-FITC Detection Kit (Roche Applied Science, Penzberg, Germany) by flow cytometer according to the manufacturer's protocol. Each experiment was performed in triplicate.