Dna content quantitation assay cell cycle kit

The cell cycle was investigated using a DNA Content Quantitation Assay (Cell Cycle) kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer's protocols. Briefly, following transfection and irradiation, cells were treated with 40X RNase A at 37°C for 20 min. Then, cells were stained with PI solution at 4°C for 15 min. Cell cycle distribution was analyzed using a flow cytometer.

PADI3-expressing HCT116 cells were prepared by the transfection of p3×FLAG-CMV-PADI3-7.1 recombinant plasmids. Cells transfected with blank p3×FLAG-CMV-7.1 plasmids were used as the negative control. Following transfection for 60 h, the cells were harvested and washed 2 times with precooled phosphate-buffered saline (PBS) and then stored at –20°C with 70% ethanol overnight. A DNA Content Quantitation Assay (Cell Cycle) Kit (Solarbio, China) was used to analyze the cell cycle. The procedure was as follows: First, overnight-fixed cells were washed twice with 1 mL of precooled PBS, and after centrifugation at 1,000 rpm for 5 min at room temperature, the cells were resuspended in 100 μL of RNase A and incubated at 37°C for 30 min. Next, 400 μL of PI solution was added to the cell-RNase A mixture and incubated at 4°C for 30 min in the dark. Finally, the cells were washed with precooled PBS and then analyzed using FCM.