Annexin 5 pe 7 aad double staining kit

The cell cycle and apoptosis analysis was described in our previous work (Liu et al., 2016 (link)). Briefly, cells were harvested and fixed in 70% ethanol for 24 h at -20°C, then treated with RNase A, and stained with 25 μg/ml of propidium iodide (PI) (Beyotime). For cell apoptosis analysis, the ratio of apoptotic cells was determined using an Annexin V-PE/7-AAD double staining kit (BD). Samples were all analyzed using a MoFlo XDP High-Performance Cell Sorter (Beckman Coulter, CA, United States), and the data were analyzed using Summit v.5.2 software according to the manufacturer’s protocol. At least three independent experiments were performed.

For cell cycle analysis, cells were harvested and fixed in 70% ethanol for 24 h at − 20 °C. The cells were then treated with RNase A and stained with 25 μg/ml propidium iodide (PI). For cell apoptosis analysis, cells were treated with serum starvation for 24 h, and then the ratio of apoptotic cells was determined using an Annexin V-PE/7-AAD double staining kit (BD Biosciences, MD, USA). Samples were all analyzed using a MoFlo™ XDP High-Performance Cell Sorter (Beckman Coulter, CA, USA), and the data were analyzed using Summit v.5.2 software according to the manufacturer’s protocol. At least three independent experiments were performed.

Apoptosis was checked using an Annexin V-PE/7-AAD double staining kit (BD Pharmingen Inc., San Diego, CA, USA) according to the manufacturer’s protocol. In short, harvested cells were cleaned by PBS and suspended in 1 × binding buffer. Aliquots of 10⁵ cells were stained with 5 μl of Annexin V/PE and 10 μl of 7ADD. Stained cells were detected via flow cytometry.