Annexin 5 pi

The effect of LPS (1.0 μg/ml) treatment BENDs on apoptosis and cell cycle was measured by flow cytometry. For cell apoptosis and necrosis, digested cells were stained by Annexin V-FITC and propidium iodide (Annexin V-PI) at 25°C for 15 min according to the manufacturer's instructions (Multisciences Biotech Co., Ltd., Hangzhou, China). For cell cycle assay, BENDs were harvested by trypsin digestion at the indicated time points (0 h, 6 h, 9 h, and 12 h) and fixed in ethanol at -20°C. Cells were then rehydrated and PI stained for 30 min at room temperature in the dark. Stained cells were examined using a flow cytometer (BD Biosciences, USA), and data were optimized using FlowJo software (Tree Star, USA).

The cell cycle phases and apoptosis rates of BMECs were determined by using flow cytometry. Briefly, BMECs were seeded in a 6-well plates, allowed to adhere, and then treated with ischemia for 48 h, followed by reperfusion for 12 h. Next, the BMECs were co-cultured with MSCs. After co-culture, the cells were harvested, washed twice with cold PBS, and resuspended in 200 μL of Annexin V/PI (MultiSciences, Hangzhou, China) for 15 min in the dark at room temperature. The cells were then analyzed by flow cytometry (BD, FACSCalibur, San Jose, CA, United States).

Cell cycle and apoptosis of HSBFs were determined using flow cytometry. Briefly, HSBFs were seeded in a six-well plate followed by treatment for 48 h. Following this, HBSFs were were harvested, washed twice with cold PBS, and resuspended in 200 μl Annexin V/PI (MultiSciences, Hangzhou, Zhejiang, China) or PI for 15 min at dark at room temperature. Following this, cells were analyzed using flow cytometry (BD, FACSCalibur, San Jose, CA, U.S.A.).