Cell cycle and apoptosis analysis 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).

Apoptotic cells were measured using Annexin V-APC/PI apoptosis detection kit (Keygen, China). Briefly, cells were washed with PBS and resuspended in 500 µl of binding buffer, and then 5 µl of Annexin V-APC and 5 µl of propylene iodide (PI) were added. After incubating the cells at room temperature in the dark for 15 min, cells were analyzed by FCM. Cell cycle was detected using cell cycle and apoptosis analysis kit (US EVERBRIGHT^® INC., Suzhou, China). Cells were digested with trypsin and washed with PBS. Subsequently, 250 μl of PBS was used to resuspend the cells, and 750 μl of absolute ethanol was used to fix the cells overnight. Before staining, the cells were centrifugated 5 min with 1,000 × g to remove the ethanol and were resuspended by 1 ml of cold PBS. Then 0.5 ml of PI was added to each sample. After incubating the cells at room temperature in the dark for 30 min, the stained cells were analyzed by FCM. FlowJo 7.6.1 software was used to analyze apoptotic rate and cell cycle.

Cell cycle analysis was performed using a Cell Cycle and Apoptosis Analysis Kit (US Everbright® Inc., Suzhou, China). Briefly, cultured cells were trypsinised to produce single cells, which were then fixed with 70% ethanol at −20 °C overnight. Cells were stained with propidium iodide for cell cycle analysis, which was performed using a cell analyser (BD LSRFortessa, BD Biosciences, San Jose, CA, USA). Data were collected and analysed using ModFit LT software (Verity Software House, ME, USA).
Apoptotic cells were quantified using the Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China). Cultured cells were digested with trypsin to produce single cells, which were then stained with annexin V-FITC and propidium iodide, according to the manufacturer’s instructions. Apoptosis analysis was performed using BD LSRFortessa analyser. Data were collected and analysed using FlowJo software (BD Biosciences).