Annexin 5 fitc pi double staining cell apoptosis detection kit

After detachment using 0.25% trypsin followed by washing with PBS, NPMSCs were resuspended in 500 µl binding buffer, and then were treated with 5 µl Annexin V-FITC and 5 µl PI. After a 15-min reaction at room temperature, the mixture was detected and analyzed using a BD FACSCalibur™ flow cytometer and BD FACSDiva™ 6.0 software (BD Biosciences). All the steps mentioned above were performed according to the instructions of the Annexin V-FITC/PI double-staining cell apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd.).

Cell apoptosis and cell cycle were respectively detected by the Annexin V-FITC/PI Double Staining Cell Apoptosis Detection Kit (KGA108; Keygen) and a Cell Cycle Detection Kit (KGA512; Keygen), in accordance to the manufacturer’s manual. Briefly, the cells were grown to 70% confluency and after 48 hours, the cells were collected and processed for analysis. The cell samples were then analyzed using a flow cytometer ACEA NovoCyte equipped with the FlowJo software [version 10; Becton, Dickinson, and Co. (BD), Franklin Lakes, NJ, USA].

DMSO and MTT were purchased from Sigma-Aldrich (Merck KGaA). Piperine (molecular weight, 285.35 kDa; National Institutes for Food and Drug Control) was dissolved in DMSO at the concentration of 150 µM and stored at −20°C. An Annexin V-FITC/PI double staining cell apoptosis detection kit was obtained from Nanjing KeyGen Biotech Co., Ltd. NQBB FBS was obtained from Wuhan ChunDuBio Co., Ltd. Anti-MMP-2 (cat. no. 10373-2-AP; 1:1,000) was purchased from ProteinTech Group, Inc. Anti-VEGF (cat. no. GB11034; 1:3,000), anti-c-Myc (cat. no. GB13076; 1:500), anti-cyclin D1 (cat. no. GB11079; 1:1,000), anti-cyclooxygenase-2 (COX2; cat. no. GB11072; 1:500), anti-β-catenin (cat. no. GB11015; 1:500) and anti-glycogen synthase kinase-3β (GSK-3β; cat. no. GB11099; 1:1,000) were purchased from Wuhan Servicebio Technology Co., Ltd.