Annexin 5 pe 7 aad kit

The percentage of apoptotic cells was determined using an Annexin V-PE/7-AAD kit (MultiSciences, Hangzhou, China) together with flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA). Cells seeded in 6-well plates were treated in culture with hesperetin (200 μM), DDP (4 µg/ml) or hesperetin + DDP (200 μM + 4 μg/ml) for 24 h. The adherent cells were collected, washed 2 times with cold PBS, and then co-stained with 10 µl 7-AAD and 5 µl Annexin V-PE in the dark for 15 min at room temperature before analysis using flow cytometry. Additionally, transfected GC cells were also exposed to hesperetin + DDP (200 μM + 4 μg/ml) for 24 h and stained as described above. Density plots were used to show four cell populations, e.g., live, early apoptotic, necrotic, and late apoptotic and dead, according to their various fluorescence characteristics: Live cells (PE and 7-AAD negative), early apoptotic cells (PE positive and 7-AAD negative), late apoptotic cells (PE and 7-AAD positive), and necrotic cells (PE negative and 7-AAD positive).

The Annexin V-PE/7-AAD kit (MultiSciences) and Annexin-V-FITC/PI kit (BD Biosciences) were used to calculate the number of apoptotic cells by flow cytometry (FACSCalibur; Becton Dickinson). All cells were cultured into six-well plates, then incubated for 24 h with different treatments. Adherent cells were collected and costained with 5 μl Annexin V-PE and 5 μl 7-AAD prior to flow cytometric analysis.

Cell cycle and apoptosis were measured using a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ). For cell cycle analysis, the cells were starved in serum-free medium for 24 h, then cultured normally for less than 24 h and harvested with tyrisin (approximately 1 × 10⁶ cells), finally fixed with 75% ethanol and stained with propidium iodide (Sigma, MO). The Annexin V PE/7AAD Kit (Multi sciences) was used for apoptosis assay. The apoptosis of cells was induced with 5-FU (1 mg/ml) and stained according to the manufacturer’s protocol. The results were analyzed using Multicycle-DNA Cell Cycle Analyzed Software.