Beyoclick edu 555 cell proliferation detection kit

Cell proliferation was assayed using the BeyoClick™ EdU-555 Cell Proliferation Detection Kit (Beyotime) according to the manufacturer’s instructions with some modifications. In brief, the cells were incubated in DMEM containing 10% FBS and 10 μM EdU (pre-balanced at 37 °C for more than 3 h) for 2 h. Next, the cells were fixed with 4% paraformaldehyde for 20 min and then permeabilized by incubation in PBS with 0.3% Triton X-100 for 20 min at room temperature. After washing three times with PBS, the cells were incubated with BeyoClick additive solution at room temperature in the dark for 30 min. Then, the cells were incubated with 10 μg/mL Hoechst 33342 at room temperature for 15 min to label the nuclei. An inverted research microscope (Ti2-U, Nikon, Tokyo, Japan) and ImageJ software (NIH, Bethesda, MD, USA) were used to analyze the number of EdU-positive cells and the total cell number. The proliferation rate was calculated as the ratio of EdU-positive cells to the total cell number.

Proliferation of RPE cells was investigated with BeyoClick™ Edu-555 Cell Proliferation Detection Kit (C0075S, Beyotime, China) according to the manufacturer’s protocols. Briefly, after being seeded into 12-well plates for 24 h at 37°C, RPE cells were incubated with Edu working solution (10 µM) for 2 h. Then washed with PBS twice and fixed with 4% paraformaldehyde for 15 min at room temperature. Next, the cells were permeabilized with 0.3% TritonX-100 (Beyotime, China) for 10 min and washed with PBS three times. Finally, cells were incubated with Click Additive Solution for 30 min in dark, and subsequently stained with 1X Hoechst for the nucleus staining. Images were captured with the fluorescence microscope (Leica, Germany). RPE cells at DNA replication phase appeared red fluorescence while the nuclei represented blue fluorescence.