Click it reaction mixture

After transfection for 48 h, Detroit562 and FaDu cells were subjected to 10 μM EdU staining solution (KeyGEN BioTECH, China) for 2 h at 37°C. after that, the cells were fixed in 4% paraformaldehyde at room temperature for 15 min, permeabilized by 0.1% Triton X-100 at room temperature for 20 min, and incubated in Click-iT reaction mixture (KeyGEN BioTECH, China) in dark at room temperature for 30 min. 6-diamidino-2-phenylindole (DAPI, Biosharp, China) was used to stain the nuclei and the target fluorescence was visualized under fluorescence microscope at 400 × magnification.

EdU staining was performed using kFluor647-EdU cell proliferation assay kit (Keygen Biotech, Nanjing, China). Forty-eight h after transfection, cells were maintained in 10 μM EdU (Keygen Biotech) for 12 h after PBS washing. Then, fixation and penetration of cells were performed, followed by cell incubation in Click-iT reaction mixture (Keygen Biotech). Hoechst 33,342 solution (Keygen Biotech) was used for DNA counter-staining. After mounting, the slices were observed and pictured under a microscope (Olympus, Tokyo, Japan) at 400x magnification.

Cells (75,000) were plated in 24-well plates with round coverslips overnight. 200 µl of EdU (10 µM, KeyGEN Bio TECH, Nanjing, China) were added to each well, and the cells were incubated at 37°C, 2 h. The cells were fixed by adding 4% neutral paraformaldehyde to each well and removed after 30 minutes at room temperature. Then, we added 0.5% TritonX-100 (KenGEN, Nanjing, China) in PBS (200 µl) to each well for 20 min and stained the samples with 200 µl Click-iT reaction mixture (KeyGEN Bio TECH, Nanjing, China) for 30 min under lightproof conditions. Next, DAPI (Beyotime, China) was used to stain the glioma cell nuclei. Round coverslips were observed using Carl Zeiss microscope (Carl Zeiss, Germany) to analyze the proportion of EdU-positive cells.