Beyoclick edu 488 cell proliferation assay kit

The EdU (5-ethynyl-2′-deoxyuridine) staining assay was performed to detect the
proliferation of HESCs. Human endometrial stromal cells (5 × 10⁴) were seeded
in a 24-well plate and cultured overnight. 500 ng/ml LPS (Sigma-Aldrich) and/or 1 μg Exos
were added onto HESCs. Twenty-four hours later, HESCs were washed with PBS once and
stained with the BeyoClick™ EdU-488 Cell Proliferation Assay Kit (Beyotime, Shanghai,
China), according to the manufacturer’s instructions. DNA staining was performed with DAPI
solution. Human endometrial stromal cells were visualized using a fluorescence
microscopy.

For the cell proliferation assay, HepG2 cells were seeded (2.5×10⁴ cells/well) into 24-well plates at 37°C for 24 h. Subsequently, cells were transfected with miR-26b-5p inhibitor for 24 h, then treated with 2 µM PPM for 37°C for 24 h. Cell proliferation was detected using BeyoClick™ EdU-488 Cell Proliferation Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Briefly, the cells were incubated with EdU solution for 2 h at 37°C, fixed with 4% paraformaldehyde for 15 min and then washed with PBS three times, both at room temperature. To perforate the cells, PBS containing 0.3% Triton X-100 was added for 10 min at 37°C, and the Click Additive solution (provided in the kit) was added and the cells were incubated at room temperature for 30 min in the dark. Nuclei were stained with 100 µl Hoechst (1,000X diluted to 1:1,000) in dark for 10 min at 37°C. After washing with PBS, cells were observed under a fluorescence microscope (Echo) at ×100 magnification, and images were analyzed using ImageJ v1.53a (National Institutes of Health).

BeyoClick EdU-488 cell proliferation assay kit (Beyotime, Shanghai, China) was used to test cell proliferation according to the manufacturer’s protocol. An inverted fluorescence microscope was used to capture images (Olympus, Tokyo, Japan). Green fluorescence indicated positive cells, whereas Hoechst-stained cells were stained with blue fluorescence.

Cell proliferation was assessed using the 5-ethynyl-2′-deoxyuridine (EdU). ICP1 cells seeded in 6-well plates were cultured to 70%–80% density and then transfected with the relevant siRNAs or overexpression vector. Forty-eight hours after transfection, the cells were fixed and stained using the BeyoClick EdU-488 Cell Proliferation Assay kit (Beyotime). An inverted fluorescence microscope (Olympus, Beijing, China) was used to capture images of cells in three randomly selected fields, and ImageJ software was used to count EdU-stained cells. The experiment was repeated three times independently.