Cd90 pe

ADSCs were characterized by flow cytometry at passage 4. Briefly, ADSCs were harvested and washed twice with phosphate-buffered saline (PBS). Then, the cells were incubated for 30 min in PBS containing anti-CD29-FITC (cat. # 555005; BD, San Diego, CA, USA), CD31-PE (cat. # 555027; BD, San Diego, CA, USA), CD49-FITC (cat. # 557457; BD, San Diego, CA, USA), CD90-PE (cat. # 551401; BD, San Diego, CA, USA), CD106-PE (cat. # 559229; BD, San Diego, CA, USA), CD34-FITC (cat. # sc-7324; Santa Cruz, Dallas, TX, USA), CD45-FITC (cat. # MCA43FT; AbD, Oxford, UK), CD73-FITC (cat. # bs-23233R; Bioss, Beijing, China), CD105-FITC (cat. # bs-10662R; Bioss, Beijing, China). The stained cells were then subjected to flow cytometry analysis.

Rat BM was isolated from male GFP transgenic Lewis rats (RRRC), as described previously [23 ].
MNC were obtained by density gradient (Hystopaque) stratification of whole BM and centrifugation at 600 × g for 30 min. The ring containing the MNC fraction was harvested, resuspended in saline containing 1 % fetal bovine serum (FBS; HyClone, South Logan, UT, USA), and washed three times (300 × g for 7 min).
MSC were isolated from whole BM by plastic adherence and in-vitro expanded as described previously [23 ]. The differentiation ability of MSC toward osteogenic and adipogenic lineages was evaluated as described previously [23 ].
MSC were characterized and analyzed for the expression of particular cell surface molecules by flow cytometry: CD45-CyChrome™, CD11b-FITC (in order to quantify hematopoietic-monocytic contamination), CD90-PE, CD106-PE, CD73-PE, and CD44-PE (BD Pharmingen, San Diego, CA, USA). 7-AAD was added to exclude dead cells from the analysis. Green fluorescence intensity was assessed by flow cytometric analysis on freshly isolated BM-MSC as well as on BM-MSC at different passages in culture. Flow cytometric acquisition for both BM-MNC and BM-MSC was performed by collecting 10⁴ events on a FACScalibur (Becton Dickinson, San Jose, CA, USA) instrument, and data were analyzed on DOT-PLOT bi-parametric diagrams using CELL QUEST PRO software (Becton Dickinson).

MC3T3-E1 Subclone 14 cells were obtained from ATCC. Cells were cultured in α-MEM culture medium (10% FBS, 100 U/mL P/S, 1% sodium pyruvate) and then removed with 0.05% trypsin-EDTA after reaching confluence at 37 °C, 5% CO₂. MSCs were isolated from Wharton’s jelly tissue from the human umbilical cord [61 (link)]. Cells were cultured in a high-glucose DMEM culture medium (10% FBS, 100 U/mL P/S, 1% sodium pyruvate) and then removed with 0.05% trypsin-EDTA after reaching confluence at 37 °C, 5% CO₂. For osteogenic differentiation, dexamethasone (0.1 μM, Sigma, USA) and ascorbic acid-2-phosphate (0.25 mM, Sigma, USA) were used.
For the characterization of MSCs, the specific surface markers of MSCs were characterized by flow cytometry. In brief, MSCs were detached, washed, and incubated with the indicated antibody conjugated with fluorescein isothiocyanate (FITC) and/or phycoerythrin (PE), against the indicated markers: CD14-FITC, CD34-FITC, CD44-PE, CD45-FITC, CD73-PE, and CD90-PE (BD Pharmingen, San Diego, CA, USA). PE-conjugated IgG1 and FITC-conjugated IgG1 were used as isotype controls (BD Pharmingen). Next, the antibody conjugated cells were analyzed by FACS analysis (LSR II, Becton Dickinson, Canton, MA, USA). MSCs in the 8th passage were used in this study.