Pi rnase staining buffer kit

Cell cycle assays were performed according to instruction for flow cytometry using the PI/RNase Staining Buffer Kit (cat #550825; BD Pharmingen). Briefly, 1 × 10⁶ cells were collected and fixed with cold 70% ethanol at − 20 °C for 24 h. The fixed cells were washed twice and stained with PI/RNase Staining Buffer for 15 min at room temperature. Next, the stained cells were analyzed using a flow cytometer (CytoFLEX S; Beckman Coulter).

For the cell cycle study, PI/RNase Staining Buffer Kit (BD, Biosciences, San Jose, CA, USA) was used. The cells were washed twice with cold PBS, fiThe in 75 % ethanol in PBS and stored at 4 °C overnight. After fiand st, ethanol was removed by centrifugation and the cells were washed twice with cold PBS and stained with 0.5 ml of PI/RNase staining buffer at 37 °C for 30 min in the dark. Analyses were performed on BD LSR flst cytometer (BD, Biosciences, San Jose, CA, USA). The experiments were repeated three times with duplicates for each treatment.

GA with 99% purity, 5-Fluorouracil (5-FU) and N-acetylcysteine (NAC) were obtained from Sigma (USA). Punicalagin, ellagic acid and punicalin with 99% purity were purchased from Chengdu Institute of biology, Chinese Academy of Sciences (Chengdu, China). CCK-8 kit was acquired from Dojindo (Japan). The 5-ethynyl-2-deoxyuridine (EdU) labeling/detection kit was purchased from Ribobio (Guangzhou, China). Annexin V-FITC apoptosis detection kit and PI/RNase Staining Buffer kit were obtained from BD Biosciences (Becton Dickinson, USA). ROS detection kit was obtained from Jiancheng Bioengineering (Nanjing, China). Mitochondrial Membrane Potential (MMP) detection kit was obtained from BestBio company (Shanghai, China). The primary antibodies against Cleaved Caspase-3, Caspase-3, P53, Bcl-2, Bax, Cyt-c, P-Akt, Akt, P-NF-κB p65, NF-κB p65, and P-IκBα were purchased from Cell Signaling Technology (CST, USA). The primary antibodies against PI3K, P-PI3K, P-IKKα, VEGF, COX-IV, and β-Actin were purchased from abcam (UK). The second antibody was obtained from LI-COR (USA). PrimeScipt™ RT reagent Kit with gDNA Eraser and SYBR@Premix Ex Taq™ II were obtained from Takara Bio (Japan). Minimum essential medium (MEM), fetal bovine serum (FBS), penicillin-streptomycin were purchased from Gibco (Grand Island, USA). Matrigel was obtained from BD Biosciences (San Jose, USA).