Mouse anti cd90

The slides of passage 3 BMSCs were fixed with 4% paraformaldehyde (Sinopharm, China) for 15 min and then rinsed 3 times using PBS. Afterwards, the cell slides were ventilated with 0.5% Triton X-100 (Beyotime, China) at room temperature for 20 min and blocked with goat serum for 30 min. A PBS wash followed each step. Diluted primary antibodies were added to the slides and coincubated at 4°C overnight. The primary antibodies used for immunostaining included mouse anti-CD90 (1/500, Abcam), mouse anti-CD11b (1/200, Abcam), mouse anti-CD44 (1/500, Abcam), mouse anti-CD29 (1/200, Abcam), rabbit anti-CD45 (1/100, Abcam), and rabbit anti-CD271 (1/500, Abcam). After 3 rinses with PBS, the slides were coincubated with diluted secondary primary antibodies (DyLight® 488-goat anti-rat IgG, 1/100, Invitrogen; DyLight® 488-goat anti-rabbit IgG, 1/100, Invitrogen) for 1 h. Following 3 times of PBS wash, the slides were coincubated with 4′,6-diamidino-2-phenylindole (DAPI) avoiding light for 5 min. After 4 subsequent PBS washes, the slides were dried using absorbent paper and mounted with an antifluorescence quenching agent (Southern Biotech, Alabama, US). Images were collected using a fluorescence microscope (Olympus BX53, Japan).

To further provide quantitative evidence of cell purity, cells were analyzed by flow cytometry. Over 1 × 10⁶ cells were collected at 48 h after the second passage for both the control and test groups. Test group Fbs and SCs were washed with PBS three times prior to overnight incubation at 4°C with the following primary antibodies: mouse anti-CD90 (1:500, Cat# ab23894, Abcam) for Fbs and rabbit anti-S100 (1:100, Cat# SAB5500172, Sigma) for SCs. In the control groups, cells were incubated with PBS under the same conditions. Both groups of Fbs were labeled with secondary antibody donkey anti-mouse IgG H&L 488 (1:500, Cat# ab150105, Abcam) and both groups of SCs with secondary antibody goat anti-rabbit IgG H&L 488 (1:500, Cat# ab150077, Abcam) for 2 h at room temperature. Samples of each group were then washed and resuspended in PBS. IgG 488-positive cells were detected using BD FACSCalibur and CellQuest software (BD Bioscience, San Jose, CA, United States), the percentage of which was calculated.