Edu 488 cell proliferation kit

The effect of exosome stimulation on chondrocytes was measured using the 5-ethynyl-2′-deoxyuridine (EdU)-488 Cell Proliferation kit (Guangzhou RiboBio Co. Ltd.) and flow cytometry, according to the manufacturer's instructions. Briefly, normal chondrocytes were seeded into 48-well plates and cultured with exosomes (400 µg/ml) for 48 h. EdU working solution, consisting of 150 µl complete culture medium for chondrocytes supplemented with 0.15 µl EdU, was added per well and incubated at 37°C for 3 h. The cells were then trypsinized, washed with PBS, fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature, neutralized with 2 mg/ml glycine for 2 h and washed again with PBS. Permeabilization was performed using 0.4% Triton X-100 for 5 min at room temperature and the cells were washed twice with PBS. The labelled chondrocytes were resuspended in the staining solution from the kit, incubated for 10 min at room temperature in the dark, washed twice with 0.4% Triton X-100 and then resuspended in PBS for flow cytometry analysis and analyzed with FACSDiva v8.0.3 software (Becton-Dickinson).

To confirm the change in the proliferation rate of the three cell types (HMEC-1, BMSCs and MC3T3-E1), an EdU-488 Cell Proliferation Kit (Ribobio, Guangzhou, China) was used and results were evaluated by flow cytometry following the manufacturer's instructions. Cells were seeded into a 48-well plate at an initial density of approximately 2 × 10⁴ cells/well, and cultured in the specified conditions for 12 h. Then, the EdU working solution, mixed with 150 μL complete culture medium and 0.15 μL EdU, was added into each well and incubated at 37°C for 3 h. After incubation, cultured cells were harvested using trypsin-EDTA, washed twice with PBS and fixed in 4% paraformaldehyde (PFA) for 10 min. The fixed cells were neutralized using 2 mg/mL glycine and washed twice in PBS. After being permeabilized with 0.4% Triton X-100 for 3 min and washed twice with PBS, the labeled cells were resuspended in Apollo staining solution (Ribobio) and incubated for 10 min. After another two washes in 0.4% Triton X-100, the prepared cells were resuspended in PBS and analyzed using a Guava® easyCyte™ flow cytometer.