EdU Proliferation Assay Protocol

The cells were incubated with 50 uM EdU solution (EdU solution and cell medium were mixed at a ratio of 1: 1000) for 2 h at room temperature. After fixation with 40 g/L paraformaldehyde for 30 min, the cells were immersed in glycine solution for 8 min. After that, the cells were permeabilized with 0.5% Triton X-100 in PBS. The cells were then stained with Apollo^® staining solution at room temperature for 30 min in the dark, and washed twice with methanol and PBS. Finally, Hoechst 33342 reaction solution (C1022, Beyotime Biotechnology Co., Ltd., Shanghai, China) was used for staining at room temperature for 20 min in the dark [44 (link)]. Three fields of view were randomly selected, and total number of EdU-stained cells, which indicates proliferating cells and Hoechst 33342-stained cells which indicates the total number of cells, were counted. Cell viability was calculated using the following formula: proliferating cells / total cells × 100%. The experiment was repeated three times.