EdU Incorporation and Visualization Protocol

To identify S-phase cells, EdU (Jena Bioscience, Jena, Thuringia, Germany) was added into culture medium at 25 μM and incubated for 30 min. Incorporated EdU was visualized by a fluorescent dye through a Cu(I)-catalyzed [3 + 2] cycloaddition (click) reaction, by which terminal alkyne group of EdU is covalently attached with azide group of a fluorescent dye, as described previously with minor modifications [39 (link), 40 (link)]. Briefly, cells were fixed with 4% paraformaldehyde (PFA) for 15 min and permeabilized with 0.5% Triton X-100 for 3 min. After three times PBS washes, cells were incubated with the reaction mixture (2 μM picolyl azide Alexa-555 (Jena Bioscience), 20 mM Sodium ascorbate (FUJIFILM Wako Pure Chemical Corporation), 2 mM 2-(4-((bis((1-(tert-butyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)acetic acid (Jena Bioscience), 1 mM CuSO4 (FUJIFILM Wako Pure Chemical Corporation) diluted in PBS) for 30 min at 37 °C. After three times PBS wash, cells were subjected to the immunostaining if necessary.