EdU Staining of ICP-1 Cells for Proliferation

EdU staining was performed using a Cell-Light EdU Apollo 488 In Vitro Kit (RiboBio, Guangzhou, China). For the EdU assay, ICP-1 cells were treated with EdU medium (1:1,000; RiboBio, Guangzhou, China) for 2 h at 37°C and then fixed in 4% paraformaldehyde solution (Yike, Guangzhou, China) for 30 min. The cells were subsequently permeabilized using 0.1% Triton X-100 solution (Gbico, New York, United States). The cells were incubated with the staining solution for 30 min at room temperature in the dark. Finally, the stained cells were scraped off with a cell scraper, collected in a 1.5 mL centrifuge tube and resuspended in 1 mL PBS. A BD Accouri C6 flow cytometer (BD Biosciences, San Jose, CA, United States) was utilized for the analysis of stained cells (16 (link)).