Cd106 pe

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).

Cultured cells were harvested, and flow cytometry was performed for phenotypic characterization. Briefly, MSCs were suspended in PBS and characterized by using the following antibodies: CD90-FITC, CD105-PE, CD106-PE, CD146-FITC, CD166-PE, CD45-APC (BD Biosciences, Le Pont-de-Claix, France), and CD73-APC, CD19-FITC, CD34-PE (Miltenyi Biotec, Paris, France). Cells incubated with phycoerythrin (PE)-conjugated (Miltenyi Biotec, Paris, France), fluorescein isothiocyanate (FITC)-conjugated, or allophycocyanin (APC)-conjugated (BD Biosciences) mouse IgG1 isotype antibodies were used as a negative control. Analyses were performed on a MacsQuant Analyser 10 (Miltenyi Biotec, Paris, France), and the results were analyzed using FlowJo software (FlowJo V10, Tree Star, Ashland, OR, USA). Phenotypic identification of cultured BM-MSCs, DP-MSCs, and WJ-MSCs are shown in Supplementary Figure S4.

The immunophenotypic characteristics of the cell cultures at P3 were determined by flow cytometry [34 ], using specific antibodies coupled with fluorochromes PE, APC, and FITC as follows: CD45-PE (554878, DB Biosciences), CD29-Biot/Strep-APC (555004, DB Biosciences), CD90-FITC (130-094-527, Miltenyi Biotec), CD71 = PE (554891, BD Biosciences), and CD106-PE (559229, BD Biosciences). Cells experimentally exposed to α-solanine expressed high levels of the CD90, CD29, CD71, and CD106 stem cell markers. The undifferentiated status of the cells was verified by inducing them to differentiate into an osteogenic lineage, according to standardized laboratory procedures (unpublished results).

Analysis of the cell surface antigen scenarios was done by using the cells obtained from the third passage. The isolated cells were incubated for 10-30 minutes in a dark environment with the following anti-human antibodies: CD90- fluorescein isothiocianate (FITC), CD 34- phycoeritrin (PE), CD 45- Peridinin chlorophyll protein (PerCP), CD 271-FITC (MiltenyiBiotec), CD106-PE, CD 44-FITC, CD73-PE, CD14-FITC (BD Biosciences), and CD105-PerCP (AbDSerotec, Kidlington, Oxford, England). Isotype-matched irrelevant monoclonal antibodies such as Mouse IgG1-PE, IgG2a-FITC (AbDSerotec, Kidlington, Oxford, England), IgG2a-PErCP, IgG2b-FITC and IgG2a-PE (Miltenyi Biotec) were used to omit the effect of non-specific cells staining. Flow cytometry analysis was done in a FACS Calibur device (BD Biosciences) by using the cell quest as data acquisition software. Win MDI software, version 8.2, was employed for data analysis.

For surface antigen detection, 5 × 10⁵ ells were washed and blocked for 1 h with PBS containing 4% FBS after detachment with 0.25% trypsin/EDTA. Cells were characterized with fluorescein isothiocyanate- or phycoerythrin-conjugated primary antibodies for human surface antigens and compared with appropriate isotype controls. The following conjugated monoclonal antibodies were used for hFOB immunophenotyping: CD90 PE (Biologend 5E10/328110), CD73 PE (Biolegend AD2/344003), CD45 PE (Biolegend HI30/304008), CD19 PE (Biolegend HIB19/302207), mouse IgG1k Isotype PE (Biolegend MOPC-21/400112), CD 44 PE (BD Bioscience 550989), CD106 PE (BD Biosciences 555647), CD34 PE (BD Bioscience 555822) and CD105 FITC (Ancell 326040), CD146 PE (Biolegend P1H12/361006). Flow cytometry analysis was performed using CytoFlex S with CytoExpert and Kaluza softwares (Beckman Coulter).

Analyses of the immunophenotype of hAdMSCs were conducted as previously described [19 (link), 20 (link)]. After being washed, hAdMSCs were subjected to incubation for half an hour at 4 °C with primary antibodies against human, including CD34-PE (cat#550761, BD Biosciences), CD44-PE (cat# 559942, BD Biosciences), CD29-PE (cat#556049, BD Biosciences), CD73-PE (cat# 550257, BD Biosciences), CD90-PE (cat# 555596, BD Biosciences), CD45-PE (cat# 560975, BD Biosciences), CD106-PE (cat# 561679, BD Biosciences), CD105-PE (cat# 560839, BD Biosciences), CD206-FITC (cat#5 51135, BD Biosciences) and HLA-DR-PE(cat# 555561, BD Biosciences). Following washing, hAdMSCs were subjected to incubation with secondary antibodies for half an hour at 4 °C. MSCs were then examined using FACSCalibur (BD Biosciences) and the FlowJo program (FlowJo, LCC). Moreover, adipogenic and osteogenic differentiation media were used to cultivate the cells. We performed oil red O staining to assess the adipogenic differentiation of cells (Solarbio, China). Alkaline phosphatase (ALP), an early osteogenic marker, and Alizarin Red (ARS), a late osteogenic marker, were used to quantify the osteogenic differentiation of the cells.