Beyoclick edu 647 cell proliferation assay kit

The EdU assay was carried out with a Beyoclick™ EdU-647 Cell Proliferation Assay Kit (Beyotime, C0081S). After treatment, the cells were immersed in EdU working solution and reaction for 2 h in a cell incubator. The EdU medium mixture was removed thoroughly, and 4% PFA in 1 × PBS was used to fix the cells for 10–15 min at RT. The fixed cells were washed 3 times with 3% BSA for 5 min, and 0.02% Triton X-100 was added to target cell, permeabilized for 10 min at RT. Click reaction solution was then added to react for 30 min at RT in the dark. After washing 3 times with 3% BSA for 5 min, Hoechst 33342 was stained the cell nucleus for 2–3 min. The immunostained cells were imaged using a microscope (OLYMPUS, IX73), and quantitative analysis of cell images was performed by ImageJ software.