Beyoclick edu 594 kit

The EdU staining was using BeyoClick™ EdU-594 Kit (Beyotime) following the manufacturer’s instructions. Briefly, 10 μM EdU was added into the fresh medium and then incubated for 2 hours at 37 °C/5% CO₂ when cells were grown to 80% confluence. After incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 20 minutes. Then the cells were incubated with reaction buffer in the dark for 30 min at room temperature. After DAPI staining, the cells were imaged with Nikon Ti2 to visualize the number of EdU-positive cells. The positive rate was determined by Image Pro Plus 6.

Cells in each group including PIK3R3 knockdown, PIK3R3 overexpression, CDKN1C+PIK3R3 knockdown, SMC1A overexpression+PIK3R3 knockdown, and control groups were prepared into single‐cell suspension and plated onto a six‐well plate at the density of 5000 cells per well. Colony formation was monitored continuously at 1, 5, and 10 days after clone establishment was confirmed at 6 h. Clone size was measured using an inverted microscope (ZEN2). For the EdU assay, cells undergoing DNA replication were detected using BeyoClick™ Edu‐594 Kit (Beyotime) according to the manufacturer's instructions.

In the cell proliferation assay, ADSCs from the same generations (between passages P4 and P6) were cultured in 96-well plates at a density of 7000 cells per well. Each experimental group consisted of five replicated wells. Following an overnight incubation, the cells were treated with the BeyoClick EdU-594 kit (Beyotime, China) for a duration of 3 h, following the manufacturer's instructions. Subsequently, the EdU-positive ADSCs (stained red) and all cells (stained blue) were visualized and quantified using a fluorescent microscope in the replicated wells of each group. The cell proliferation rate was determined by calculating the ratio of the number of red-stained cells to the number of blue-stained cells, multiplied by 100%.
In the cell migration assay, ADSCs from the same generations (between passages P4 and P6) were cultured in the upper chamber of a 24-well transwell insert (8.0μm pore size, Corning, AZ, USA). The seeding density was 3×10⁴ cells per well, using Dulbecco's modified Eagle medium without fetal bovine serum. The lower chamber was filled with 650μl of ADSCs medium. After 24 h of culture, the cells that migrated to the lower chamber were stained with crystal violet and visualized using a microscope. The number of migrated cells was then quantified for each group.

The EdU Assay was used to detect newly synthesized DNA to measure cell proliferation. After incubating cells in culture medium with EdU (Beyotime Biotechnology, Shanghai, China) for 1/10‐1/5 cell cycle, cells were fixed then stained with the BeyoClick EdU‐594 Kit (Beyotime Biotechnology, Shanghai, China) and DAPI. A Cytation 5 was used to obtain pictures.