Cell light edu fluorescence detection kit

A Cell-Light EdU fluorescence detection kit (Guangzhou RiboBio Co., Ltd.) was used to detect EdU-positive cells according to the manufacturer's instructions. The cells were seeded into 96-well plates (5,000 cells/well), added with 100 μl medium containing 50 μmol/l EdU, and cultured at 37°C with 5% CO₂ for 2 h. The cells were then washed with PBS 3 times and each well was supplemented with 100 μl PBS solution containing 4% paraformaldehyde to fix the cells at room temperature for 30 min. Following PBS washing, each well was supplemented with 100 μl PBS solution containing 0.5% TritonX-100 to increase membrane permeability. Following PBS washing again, each well was added with 100 μl Apollo^® staining reaction solution (provided with the kit) and cultured at room temperature in the dark for 30 min. The staining reaction solution was discarded and each well was supplemented with 100 μl DAPI (C1002, Beyotime Institute of Biotechnology, Inc.) at room temperature in the dark for 30 min. Following PBS washing, the staining effect was observed under fluorescence microscope (FSX100, Olympus Corporation) and images were collected. The images were analyzed using Olympus stream system. The total number of nuclei (blue) and proliferating cells (red) were counted respectively. EdU-positive cell rate=proliferating cells/total cells ×100%. The experiment was repeated three times.

The DNA replication ability of cells was measured in line with the instructions of a Cell-light EdU fluorescence detection kit (RiboBio, Guangzhou, Guangdong, China). Five visual fields were randomly photographed under the fluorescence microscope (Olympus FSX100). Blue fluorescence represented all cells, while red fluorescence represented the replicating cells infiltrated by EdU. The percentage of EdU-positive cells was then calculated.