Cd105 pe

hBM-MSCs or hfC-MSCs were stained with following anti-human monoclonal antibodies labelled with Fluorescence isothyocyanate (FITC) or phycoerythrin (PE): CD73-PE, CD90-PE, CD105-PE, CD117-PE, SSEA-4-PE, CD31-FITC, CD34-FITC, CD45-FITC, HLA-DR-FITC (all from Serotec), or isotype matched control monoclonal antibodies (Becton Dickinson). Stained cells were analyzed in Flow Cytometer (FACS Calibur, Becton Dickinson).

The following monoclonal antibodies were used for the characterization of MSCs: CD45-FITC (BD Biosciences clone HI30, dilution 1:100), CD34-PE (BD Biosciences clone 581, dilution 1:5), CD73-PE (BD Biosciences clone AD2, dilution 1:20), CD90-PE (BD Biosciences clone 5E10, dilution 1:100), MHCI-PECy7 (BD Biosciences clone G46-2.6, dilution 1:20), CD80-PECy7 (BD Biosciences clone L307.4, dilution 1:20), CD105-PE (Serotec clone SN6, dilution 1:10), MCHII-FITC (Biolegend clone L243 at 1:200), and CD86-AF488 (Biolegend clone IT2.2, dilution 1:20). For staining, the cells were washed with FACS buffer (PBS containing 1% FBS and 0.1 % NaN₃). The antibodies were diluted in 50 μL of FACS buffer, combined with 1 × 10⁵ cells/sample and incubated for 30 min at 4 °C. The unbound antibody was removed by washing twice with FACS buffer. The cells were resuspended in FACS buffer for analysis on a BD FACS Canto. Compensation parameters were established on the FACS Canto using appropriately single stained cells or compensation beads and fluorescence minus one (FMO) controls. Propidium iodide (PI) was used as a viability dye to exclude dead cells and debris from analysis. The data were analyzed using the FlowJo software version 10 (Tree Star Inc, OR, USA) .

Colonies expanded from blood were trypsinised, washed and stained with antibodies: CD31-FITC, CD90-PE, CD105-PE (AbD Serotec, Kidlington, UK), CD19-PE, CD33-FITC, CD34-PerCp, CD45-PE-Cy7, CD61-FITC, CD73-PE, (BD Biosciences, Oxford, UK) and CD271-APC (Miltenyi Biotec), at manufacturers recommended concentrations. Cells were washed, 30,000 events captured on a LSRII flow cytometer and the data analysed using FACSDiva Software (both BD Biosciences).

To prove cultures' MSC identity p1 and p3 cultures were trypsinised and stained with the following antibodies: CD90-PE, CD105-PE, CD31-FITC (all from Serotec, Kidlington, UK), CD73-PE, CD34-PerCp, CD45-PE-Cy7, CD19-FITC, CD14-PE, CD146-PE, and HLA-DR-FITC (all from BD Biosciences, Oxford, UK). All antibodies were used at manufacturers' recommended concentrations and the data were collected and analysed using a LSRII flow cytometer equipped with FACSDiva Software (both from BD Biosciences).