5 ethynyl 2 deoxyuridine edu cell proliferation assay kit

For in vitro cell viability assay, GDF11-overexpressed and negative control-MSCs were plated on 96-well plates (2 × 10³ cells/well) and cultured in serum-free medium under hypoxia (0.1% O2, 5% CO2) at 37 °C for 48 h. For control, MSCs were plated in complete culture medium with normoxia (21% O2, 5% CO2) condition for the same period. Then, 10 μl of Cell Counting Kit-8 (CCK-8, Dojindo, Japan) in 90ul medium was added and incubated for 2 h at 37 °C, and the absorbance was determined at a wavelength of 450 nm. MSC viability was evaluated using OD value as described above. The cells proliferation was also measured using the 5-ethynyl-2′-deoxyuridine (EdU) Cell Proliferation Assay Kit (Ribobio) according to the manufacturer’s instructions. EdU-labeled cells were counted manually in five fields of view randomly selected from each well, and percentages were calculated.

Cell proliferation was analysed by 5‐ethynyl‐2′‐deoxyuridine (EdU) cell proliferation Assay Kit (Ribobio, Guangzhou, China). In brief, cells were cultured in 48‐well plates for 12 hours and then transfected with Si‐XIST or Si‐NC. After another 12 hours, Si‐XIST or Si‐NC silenced cells were infected with inactive E. coli or S. aureus for 24 hours. Then, the cells were incubated with EdU solution for 2 hours at 37°C. The labelled cells were fixed with 4% paraformaldehyde for 20 minutes and then incubated with glycine. Subsequently, cells were treated with 0.5% Triton‐X100 for 10 minutes and washed with PBS. After that, cells were stained with Apollo for 30 minutes in dark. Followed by staining with Hoechst‐33342 for another 30 minutes, the proportion of cells was finally shown as the ratio of the fluorescent positive cells to total labelled cells.

SCs were seeded on 96-well plates at a density of 2 × 10⁴ cells/mL overnight. EC-EXO of different concentrations (1, 10, 50 and 100 μg/mL) were added to each well and cultured for 24 h and 48 h. Then 10 μL of Cell Counting Kit-8 (Dojindo, Japan) was added into each well and incubated for 2 h at 37 ℃ in humidified air containing 5% CO₂. The cell proliferation was determined using a full-wavelength microplate reader at 450 nm. We also used the CCK8 assay to study the growth curve of SCs in different groups. Furthermore, the 5-ethynyl-2ʹ-deoxyuridine (EdU) Cell Proliferation Assay Kit (Ribobio) was also used to measure the cell proliferation of SCs in different groups. EdU-labelled cells were counted manually in three fields of view randomly chosen from each well to calculate the percentages. To study the clonogenic ability of SCs in different groups, cells with different treatments were seeded to 6-well plates (1000 cells/well). SCs were cultured for 10 days and stained with 0.1% Crystal Violet Stain solution (Solarbio). The numbers of colonies were counted manually, and the inverted microscope detected the morphology of the colonies from different groups.