Dapi staining solution dapi

Cells were collected and fixed in 70% ethanol at 4°C overnight. Then, the cells were treated with RNase A (50 μg/mL, Fuyuanbio, China) and stained with DAPI Staining Solution (DAPI, 10 μg/mL, Beyotime, China) for 30 min at 37°C after washing with PBS. The distribution of the cell cycle phases was calculated by a FACSCalibur flow cytometer (Becton, Dickinson and Company, CA). The phase ratio (%) was regarded as the percentage of cells in the G1/S/G2 phase. Each experiment was conducted in triplicate.

BMSCs and tBMSCs that were growing logarithmically were adjusted to a density of 5×10⁴ cells/ml and cultured on cover slips overnight. Then, cells were fixed in 4% paraformaldehyde for 20min, permeabilized in 0.5% Triton X-100 at room temperature for 20min and incubated with 0.3% H₂O₂ at 37°C for 30min. Then, the cells were blocked with 5% serum at 37°C for 20min followed by incubation at 4°C overnight with the following primary antibodies from Abcam, UK: (1) CD105, 1:200, clone number 8A1; (2) CD90, 1:200, clone number IBL-6/23; (3) CD44, 1:150, clone number T2-F4; (4) CD29, 1:150, clone number KM16; (5) CD45, 1:200, clone number IBL-3/16; (6) CD34, 1:200, clone number ICO-115; (7) CD-11b, 1:200, clone number EPR1344, and (8) CD31, 1:200, clone number: JC/70A. Next day, after washings with immune staining solution (P0106C, Beyotime, Shanghai, China), the cells were incubated with secondary antibodies of Cy3-labeled Goat Anti-Rabbit/Rat IgG (H+L) (1:500, Beyotime, Shanghai, China) at 37°C for 1h followed by staining with DAPI Staining Solution DAPI (C1005, Beyotime, Shanghai, China) for 3-5min. Finally, they were mounted in fluorescence quenching solution and observed under a fluorescence microscope (MF53, Mshot, Guangzhou, China).