Facs dive software version 6

To separate BMSC from the medullary cavity, female mice were sacrificed, and BMSC were washed from the femora and treated at 4 °C for 20 min with FITC, PE, and allophycocyanin conjugated antibodies, as well as peridinin chlorophyll protein conjugated to CD29, CD45, CD11b, and Sca1 (BioLegend). Acquisition and analysis were carried out using the FACS Aria model and FACS DIVE software version 6.1.3 (BD Biosciences). Primary BMSC were isolated and planted in culture flasks for cell population enrichment. They were subcultured about 1 week later, when the secondpassage BMSC clustered. The osteogenesis induction media (5 mmol/L glycerophosphate and 50 g/mL ascorbic acid, 300 ng/mL BMP2) was applied to BMSC for 48 h in 24-well plates (5 × 10⁵ cells/well). Then, homogenize the cell lysates to determine ALP activity using the enzymatic colorimetric ALP Kit (from Roche) and spectrophotometric determination of the output of pnitrophenol. The amounts of secreted osteocalcin were determined in culture media using an immunoassay kit (DiaSorin).

We isolated primary BMSCs as reported previously.26, 27 We collected all bone marrow cells and incubated them with FITC‐, APC‐ and PE‐conjugated antibodies which recognized mouse Sca‐1 (BioLegend, 108108), CD29 (BioLegend, 102206), CD45 (BioLegend, 103132) and CD11b (BioLegend, 101226) for 30 minutes at 4°C. Then, we performed fluorescence‐activated cell sorting (FACS) and analysis the results using FACS DIVE software version 6.1.3 (BD Biosciences). The sorted mouse Sca‐1⁺CD29⁺CD45⁻CD11b⁻ BMSCs were cultured with α‐MEM (Gibco‐BRL Co.) supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin.

We isolated and collected BMSC of mouse as before, as well as the cultivation.18 In order to isolate BMSC from medullary cavity, female mice were killed, and BMSC were washed out from femora and incubated at 4°C for 20 minutes with FITC‐, PE‐ and allophycocyanin‐conjugated antibodies, and peridinin chlorophyll‐protein that combined with CD29, CD45, CD11b and Sca‐1 (BioLegend). For the isolation of human BMSC, we use the same method to harvest human BMSC. The human BMSC were incubated at 4°C for 30 minutes with antibody of allophycocyanin‐, FITC‐ and PE‐conjugated which recognized CD45, CD146 and STRO‐1 (BioLegend). By using FACS Aria model and FACS DIVE software version 6.1.3(BD Biosciences), acquisition was carried out and the analysis was enforced.
Here, we find out that the mouse BMSC were sorted as CD29+Sca‐1+CD45−CD11b‐, while human BMSC (hBMSC) were sorted as CD146+STRO‐1+CD45‐. Then, we gathered and cultured them for 1‐2 weeks. In culture flasks, the primary BMSC were separated and seeded for cell population enrichment. Approximately 1 week later, as the second‐passage BMSC reached clustered, they were subcultured. Afterwards, we induced adipogenic and osteogenic differentiation in the third‐passage BMSC. Plasmid transfection also executed in third‐passage BMSC.