Keyfluor488 edu kit

The proliferation of cells was determined using the EdU staining proliferation kit (KeyFluor488 EdU Kit, keyGEN BioTECH, Jiangsu, China), following the manufacturer's instructions. Cells were seeded in 96-well plates (1 × 10⁴ cells/well) in 100 μL medium. The next day, the cells were cultured with 20 μM EdU for 2 h under 5% CO₂ and 37°C. Then, 4% paraformaldehyde was added to fix the cells for 30 min, and 0.5% Triton-X-100 in PBS was added and incubated for 20 min. After the removal of the solution, 1x Click-iT® reaction cocktail was added to cells and incubated for 30 min. The staining cocktail was removed and cells were counter-stained by 500 nM DAPI in PBS and the images were captured under a Leica AM6000 microscope (Leica, Wetzlar, Germany).

DNA synthesis was monitored via EDU incorporation using commercial KeyFluor488 Edu Kit (keyGEN Biotech, Jiangsu, China). 4,6-diamino-2-phenyl indole (DAPI) was used to mark cell nucleus. The fluorescence images were captured using the fluorescence microscope (Olympus, Tokyo, Japan). The relative rate of EDU incorporation was analyzed.

EdU assay was conducted to analyze cell proliferation ability using KeyFluor488 Edu Kit (keyGEN Biotech, Jiangsu, China). Chondrocytes were seeded onto 24-well plates at 5 × 10⁴ cells/well. Cell nucleus was stained with 4,6-diamino-2-phenyl indole (DAPI; Sigma). The fluorescence intensities were determined under the fluorescence microscope (Olympus, Tokyo, Japan). The representative images were captured at the magnification of 100×.