Dna content quantitation assay kit

BMSCs and hAMSCs at different cell cycle stages were detected via the DNA content quantitation assay kit (Solarbio). They were digested with trypsin, collected, and then blended with pre‐cooled 70% ethanol and incubated overnight. The cells were washed with D‐PBS, then mixed with RNase A, and incubated at 37°C for 30 min. Subsequently, the cells were mixed with propidium iodide staining solution and incubated in darkness at 4°C for 30 min. Finally, flow cytometry (BD Biosciences) was used for analysis.

After 48 h of transfection, LX-2 cells were subjected to detection of cell cycle using the DNA Content Quantitation Assay Kit (CA1510; Solarbio, China). Concretely, the cells were digested by trypsin (EDTA 0.25%) (25200056; Gibco, USA), and resuspended with DMEM. Then 70% ethanol (48075; Merck, Darmstadt, Germany) was added to treat the cells at 4°C for 12 h, followed by washing with pre-cooled PBS. Subsequently, the cells (1 × 10⁶ cell/mL) were reacted with 100 µL RNase solution in a bath at 37°C for 30 min, and then were mixed with 400 µL propidium iodide solution at 4°C in the dark. After 30 min, the cell cycle of the cells was analyzed by a flow cytometer (CytoFLEX, Beckman Coulter, Brea, CA, USA) at an excitation wavelength of 488 nm.

A DNA Content Quantitation Assay kit (Solarbio, CA1510) was employed to detect cell cycle. Briefly, the cells were cultured in different medium for 72 h and fixed in 70% ethanol overnight. The suspensions were digested with 100 μl RNase A (30 min, 37 °C), and subsequently labeled by 400 μl propidium iodide (PI) (30 min, 4 °C, preserved in dark place). The PI fluorescence of each group was measured by flow cytometer (BD FACS Calibur TM, Becton-Dickinson, USA), and the percentage of cells in each phase was calculated.

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.