Annexin 5 pe apoptosis assay kit

Poly-2-hydroxyethyl methacrylate (poly-HEMA, Sigma, USA) was prepared in anhydrous ethanol at a concentration of 10 mg/mL. Next, 1.5 mL of the poly-HEMA solution was added to a 6-well plate and then dried for 24 h under sterile conditions. The wells coated with poly-HEMA were immediately exposed to ultraviolet light for disinfection and washed twice with sterile PBS. Approximately 5 × 10⁵ cells in a single-cell suspension were added to each well and cultured at 37 °C with 5% CO2 for 48 h. The cells were then collected and washed twice with sterile PBS. Flow cytometry (FCM) was used to detect the incidence of anoikis among cells stained with an Annexin V-PE apoptosis assay kit (KeyGEN BioTECH, KGA108, Nanjing, China).

The cell cycle distribution of CRC cells was measured by DNA Content Quantitation Assay kit (Solarbio, Nanjing, China). CRC cells were cultured in DMEM medium without FBS to synchronize them with the G1 phase for 24 h. Following the removal of the FBS-free medium, the cells were treated with 0, 12.5, 25, and 50 μM of ST in DMEM medium contained 10% FBS for 48 h. CRC cells were soaked overnight in 70% ethanol at − 20°C environment in order to immobilization. Immobilized cells were washed twice with PBS and stained with propidium iodide element. For apoptosis assay, a 48-hour treatment of CRC cells with varying concentrations (0, 12.5, 25, and 50, M) of ST in DMEM with 10% FBS was conducted. Afterward, cells were collected and apoptosis rates were measured using the Annexin V-PE apoptosis Assay Kit (KeyGen BioTECH, Jiangsu, China). Flow Jo (version 7.6.1) was used for analysis of the flow cytometry results.