Yf647a annexin 5 and pi apoptosis kit

AL (dihydrochloride form; purity > 99%) (Jiangsu Chia-tai Tianqing Pharmaceutical Co., Ltd. (Nanjing, China) was dissolved in double-distilled water to various concentrations for oral and intra-tumor administration in mice. Sodium HA (purity >95%, MW, 90 kDa), tyramine hydrochloride (Tyr·HCl), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC·HCl), hydrogen peroxide (H₂O₂, 30 wt.%), horseradish peroxidase (HRP, 100 U/mg), and bovine testicular hyaluronidase were purchased from MeiLun Co., Ltd. (Dalian, China). Dimethyl sulfoxide and crystal violet were purchased from Kelong Co., Ltd. (Chengdu, China). Polyclonal antibodies against Ki-67 and VEGF-A were purchased from Bioworld Technology Co., Ltd. (Nanjing, China). The Cell Cycle and Apoptosis Analysis Kit and YF647A-Annexin V and PI Apoptosis Kit were purchased from US Everbright, Inc. (Nanjing, China).

EdU (5-ethynyl-20-deoxyuridine) incorporation assay was performed using an EdU assay kit (YF®647A Click-iT EdU Imaging Kits; US Everbright, China) following the manufacturer's guidelines. Briefly, cells were incubated with 10 µM EdU and subsequently fixed in 4% paraformaldehyde. After EdU staining, cell nuclei were stained with DAPI, and cell proliferation was detected by a BD FACS Canto™ System (BD Biosciences, USA).
DNA content analysis was performed using a cell cycle analysis kit (Sangon Biotech, China) following the manufacturer's guidelines. Briefly, cells were dissociated by trypsin and fixed with chilled 70% ethanol overnight. The staining working fluid of propidium iodide (PI) and RNase A was used to stain DNA for 30 min. Cells were washed and filtered through a 40 µm cell strainer before flow cytometry. Cell cycle distributions were then analyzed by a BD Accuri™ C6 Plus flow cytometer (BD Biosciences, USA).
Cell apoptosis was detected using YF®647A-Annexin V and PI Apoptosis Kit (US Everbright, China) following the manufacturer’s instructions except that the cell nuclei staining dye was changed from PI (supplied with the kit) to DAPI (Beyotime, China). After staining, the activity of Annexin V/DAPI was then examined using a BD FACS Canto™ System (BD Biosciences, USA).