Keyfluor488 click it edu kit

KeyFluor488 Click-iT EdU kit (KeyGen BioTECH, Nanjing, China) was used to detect morphological changes that occurred after cells were treated with SB. Nuclear was stained with Hoechst 33342 (1 μg/mL) for 15 min at room temperature (RT). After washing by PBS, samples were visualized at 40× magnification (Olympus IX53, Tokyo, Japan).

An EdU assay was used to evaluate cell proliferation capacities by investigating the rate of DNA synthesis in cells. YMSCs and AMSCs were seeded in a six-well plate (2 × 10⁵ cells per well) and cultured for 4 days until the cells reached 70–80% confluence before the 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, which was performed with a keyFluor488 Click-iT EdU kit (keyGEN BioTECH, Nanjing, China) according to the manufacturer’s protocol. EdU-positive cells were visualized with an inverted fluorescence microscope (Olympus, Tokyo, Japan).

KeyFluor488 Click-iTEdU kit (KeyGEN, Nanjing, China) was used for analysis of cell proliferation [35 (link)]. ESCs were seeded in 96-well plates (5 × 10³ cells/well). Forty-eight hours later, EdU labeling media (50 μM) was added to the plates and maintained for 2 h under 5% CO₂ at 37°C. Then the cells were fixed with 4% polyformaldehyde containing PBS. DAPI was used to label cell nuclei. Images were observed using a fluorescence microscopy (Nikon).

Cell proliferation was also determined by 5-Ethynyl-2’-deoxyuridine assay using a KeyFluor488 Click-iT EdU kit (KeyGEN BioTECH, Nanjing, China). The EdU assay was performed according to the manufacturer’s instruction. The cells were then visualized under a fluorescence microscopy (20 × 10). To assess cell proliferation activity, the ratio of EdU-stained cells (with green fluorescence) to Hoechst-stained cells (with blue fluorescence) was calculated. Experiments were repeated at least three times.

Cell proliferation was analyzed with EdU assays. Cells were cultured in 96-well plate and treated with 100 μL of medium containing 20 μM EdU. After incubation at 37 °C, with 5% CO₂ for 2 h, the cells were fixed with 4% paraformaldehyde for 30 min and incubated with 0.5% Triton-X-100 in PBS for 20 min. The nuclei were stained with DAPI. The rate of proliferation was calculated according to the manufacturer’s instructions (KeyFluor488 Click-iT EdU Kit, keyGEN BioTECH, Jiangsu, China). Images of five randomly selected areas of each group were taken with a fluorescence microscope (Leica, Wetzlar, Germany).

Cell counting Kit-8 (CCK8) and EdU assay were used to investigate proliferation. We used a CCK-8 kit (Dojindo, Shanghai, China) to measure proliferation of cells. MSCs and im-hMSCs were cultured to P10. Then, 100 μl of cell suspension containing 1000 cells was added to each well in a 96-well plate and placed in a 37°C incubator containing 5% CO₂. After the cells were attached, a mixture of 10 μl of CCK-8 reagent and 90 μl of medium was added to each well of a 96-well plate, and five replicate wells were repeated. Incubate in a 37°C incubator containing 5% CO₂ for 1 to 2 hours, then test the OD450 with a microplate reader (Biotex, USA).
In addition, the cells were cultured in 96-well plates and treated with 100 μl of medium containing 20 μM EdU (5-ethynyl-20-deoxyuridine, Keygene, China). After incubating for 2 hours at 37°C containing 5% CO₂, the cells were fixed with 4% paraformaldehyde for 15 minutes. Cell nuclei were stained with DAPI. The rate of proliferation was calculated according to the manufacturer's instructions (KeyFluor488 Click-iT EdU Kit, keygen BioTECH, Jiangsu, China). Finally, images were obtained with an inverted fluorescence microscope (Leica, Germany).