Annexin 5 apc

In brief, the bladder cancer cells or epithelial cells were first centrifuged, resuspended and subsequently stained with Annexin V-APC (5 µL) and/or propidium iodide (PI, 5 µL) (Sigma). The CytoFLEX flow cytometry machine (Beckman, Shanghai, China) was then employed to assess cell apoptosis or cell cycle progression.

Apoptosis of GCs was evaluated using flow cytometric assay. In brief, GCs were collected and stained with Annexin V-APC and PI (Sigma Aldrich, St. Louis, MO, United States of America) at room temperature for 20 min in the dark. The fluorescence of the cells was measured by flow cytometry (BD FACSVerse, Waltham, MA, United States of America).
The level of reactive oxygen species (ROS) was detected using the Reactive Oxygen Species Assay Kit (S0033, Beyotime, Shanghai, China) according to the operating manual. The fluorescence of the cells was measured by flow cytometry (BD FACSVerse).

The following reagents and instruments were used in this study: dual-luciferase reporter assay system (Promega Madison, US), fetal bovine serum (FBS), Dulbecco's modified eagle medium (DMEM) cell culture medium (Gibco, Rockford, US), radio immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China), Matrigel (BD, New Jersey, US), cell counting kit-8 (CCK8) assay kit (Dojindo Corp, Kyushu, Japan), Gene Mutation Kit and SYBR Green Premix Ex Taq™ II (TaKaRa, Dalian, China), Pierce^TM Magnetic RNA-Protein Pull-Down kit, Lipofectamine 3000, M-MLV reverse transcriptase kit, miRNA reverse transcriptase kit, TRIzol reagent, and SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, Inc., Waltham, US). Antibodies were purchased from Santa Cruz (Dallas, US). Propidium iodide and APC-Annexin V were purchased form Sigma-Aldrich (St. Louis, USA). The PsiCHECK™-2 vector was purchased from Promega (Madison, US).

Propidium iodide, APC-Annexin V and protease inhibitor cocktail were provided by Sigma (St. Louis, MO, USA); CCK-8 test kit was provided by Dojindo Corp (Kyushu, Japan); lipofectamine 3000 and Trizol were from Thermo Fisher Scientific (Waltham, MA, USA); Apoptosis Detection Kit was from Beyotime (Nanjing, China); Dual-Luciferase Reporter Assay System was provided by Promega (Madison, WI, USA); matrigel was from BD (New Jersey, USA); Macoy’s 5A medium was from Gibco (Rockford, MD, USA); SurePrep™ Nuclear or Cytoplasmic RNA Purification Kit was from Fisher BioReagents^® (Fair Lawn, NJ, USA). Antibodies against the Bcl-2 (# 3498), GAPDH (#2118) and Cleaved caspase3 (# 9661S) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA); antibody against the DACT1 was from Novus Biologicals, LLC (Centennial, CO, USA).