Edu cell proliferation detection kit

The proliferation capacities of OS cells were measured using the EdU Cell Proliferation Detection Kit (Beyotime, Shanghai, China). 48 hours after transfection, OS cells were co-incubated with 500μL Click Reaction Buffer and were incubated for approximately 30 min. To measure the percentage of cell proliferation, Hoechst 33342 were employed for nuclear staining. Images were captured utilizing a fluorescence microscope. Representative images were selected at least three random fields of view.

SMCs were seeded onto 24-well plates at a density of 2 × 10⁴/well in triplicate. The cells were then starved prior to intervention and divided into groups, as follows: controls; SMCs stimulated with 10 ng/mL PDGF-BB (PDGF); SMCs treated with 10 µM Rosuvastatin (Rosu); and SMCs co-treated with 10 ng/mL PDGF-BB and 10 µM rosuvastatin (PDGF + Rosu). Each group was cultured for 24 h, after which an EdU cell proliferation detection kit (Beyotime) was used to evaluate the proliferation rate according to manufacturer instructions. The cells were counterstained with Hoechst-33342 (Beyotime) for 10 min and then observed with a confocal fluorescence microscope (Carl Zeiss). Hoechst-33342 and EdU staining showed blue and green fluorescence, respectively. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to count Hoechst-33342- and EdU-labeled cells, and the EdU/Hoechst-33342 ratio was then calculated.

An EdU cell proliferation detection kit (Beyotime, China) was used to detect cell proliferation. The NPMSCs were seeded into 6-well plates at a density of 4 × 10⁴ ~ 5 × 10⁴ cells/well at 37°C under 5% CO₂. After incubated with EdU for 2 h, the NPMSCs were fixed with 4% paraformaldehyde for 15 min, permeated with 0.5% Triton X-100 for 10 min, and incubated with Click Reaction Mixture for 30 min, successively. The cells were then washed with PBS and counterstained with Hoechst 33342 in the dark. A fluorescence microscope and ImageJ software (NIH, USA) were used to determine and analyze the number of EdU-positive nuclei.

EdU cell proliferation detection kit (Beyotime, China) was used to detect the proliferation of NP cells. The NP cells were seeded in a 24-well plate with cell slides. After the cells reached the target confluence, the preheated EdU working solution was added to the culture medium, and the incubation continued for 4 hours. Then, the cell slide was taken out, fixed with 4% paraformaldehyde for 15 minutes at room temperature, washed with PBS solution containing 3% BSA, and then treated with 0.3% Triton for 10 minutes. We incubate the cell slide with click additive solution for 30 minutes in the dark at room temperature. Then, the nuclei were stained with Hoechst, and images were collected using a fluorescence microscope (Leica, Germany).

Cells were seeded at a density of 1 × 10⁵ in 24-well plates including a total of 6 groups with 3 replicates each. These 6 experimental groups were cells expressing si-PIK3R3, cells expressing a NC for the si-PIK3R3, cells expressing a miR-27a mimic, cells expressing a NC for the miR-27a mimic, cells expressing a miR-27a inhibitor and cells expressing a NC for the miR-27a inhibitor. After the cells were cultured overnight, they were labeled with EdU according to the instructions of the EdU cell proliferation detection kit (Beyotime, Shanghai, China). Then, the cells were incubated at 37 °C, 5% CO₂ for 9 h before fixation and staining in the dark. The results were observed and photographed with a fluorescence microscope.
For CCK-8 assays, cells were seeded in 96-well plates at a density of 1 × 10⁴ with 100 μL of complete medium. The cells were transfected as described above, with each condition replicated 6 times. Following transfections, the cells were cultured at 37 °C, 5% CO₂ for 48 h. Following this incubation, the cell culture medium was replaced, 10 μL of CCK-8 reagent was added to each well (Yeasen, Shanghai, China) and the cells were returned to the CO₂ incubator. After 4 h, the absorbance value at a wavelength of 450 nm was measured using a microplate reader.

Cell proliferation was assessed using the EdU cell proliferation detection kit (C0071S, Beyotime, Shanghai, China). Differently-treated cells (5000 cells/well) were cultured in 96-well plates for 24 h, supplemented with a final concentration of 50 µmol/L EdU solution per well, and incubated at 37 °C for 2 h. Then, cells were fixed at room temperature with 4% paraformaldehyde for 15 min, incubated in the dark with Apollo fluorescent staining solution for 30 min, washed twice with methanol, and stained with DAPI staining solution for 30 min at room temperature, followed by three rinses with phosphate-buffered saline (PBS). The total number of cells and the number of EdU positive cells from 5 fields randomly selected under a fluorescence microscope (Leica, Germany) were applied for calculation of the percentage (%) of EdU positive cells.