Cd105 pe clone sn6

Multiparameter analyses of stained cell suspensions were performed on FACS CANTO II (BD Bioscience) and analyzed with FlowJo v10.4 software (Treestar).
To quantify the level of TNAP in BMSCs after co-culture with AML or ALL cell lines, primary AML cells, or healthy CD34⁺ progenitors, the adherent layer was subjected to trypsinization after washing with PBS to remove non-adherent cells. Cells were stained with TNAP-PE (clone W8B2; Biolegend) and with a fluorochrome-conjugated mAb specific to exclude residual leukemia cells. Specifically, CD33-PE-Cy7 (clone P67.6; BD Bioscience) was used to gate AML cells, CD19-PE-Cy7 (clone J3-119; Beckman Coulter) was used to gate B-ALL cells and CD34-FITC (clone 581; BD Pharmingen) was used to gate CD34⁺ progenitors.
The following antibodies were used for the analysis of BMSCs: CD90-PE (clone 5E10; eBioscience), CD73-PE (clone AD2; BD Pharmingen), CD105-PE (clone SN6; eBioscience), CD146-PE (clone P1H12; BD Pharmingen), CD45-FITC (clone HI30, BD Pharmingen), and CD34-FITC.
Median fluorescence intensities were calculated in co-cultured BMSCs in comparison to control with FlowJo software.

The expanded MSC population at third passage in cell culture were characterized by morphology (spindle-shaped cells) and by immunophenotypic analysis for the expression of the following membrane markers: mouse anti-human CD14-PE (clone 61D3), CD29-FITC (clone TS2/16), CD90-FITC (clone eBIO5E10) and CD105-PE (clone SN6) (from eBioscience), CD31-FITC (clone WM59), CD34-FITC (clone 8G12), CD45-PerCP-Cy5.5 (clone MOPC-21) and CD146-PE (clone P1H12) (from BD Biosciences, San Jose, CA, USA). Detection of osteocalcin in MSC culture by flow cytometry was performed with mouse anti-human PE-conjugated monoclonal antibody (R&D system) or isotype control antibody according to the manufacturer’s protocol.

The MSC population expanded in the third passage in cell culture was characterized by morphology (spindle-shaped cells) and by immunophenotypic analysis for the expression of the following membrane markers: mouse anti-human CD14-PE (clone 61D3), CD29-FITC (clone TS2/16), CD90-FITC (clone eBIO5E10), and CD105-PE (clone SN6) (from eBioscience), CD31-FITC (clone WM59), CD34-FITC (clone 8G12), CD45-PerCP-Cy5.5 (clone MOPC-21), and CD146-PE (clone P1H12) (from BD Biosciences, San Jose, CA, United States). The detection of osteocalcin in MSC cultures by flow cytometry was performed with mouse anti-human PE-conjugated monoclonal antibody (R&D system) or control isotype antibody according to the manufacturer’s protocol.