Cd166 pe

For phenotypic characterization of MSCs, monoclonal antibodies conjugated with different fluorochromes (Fluorescein [FITC]/Phycoerythrin [PE]/Alexa-647 [AL-647]), which combine a number of both positive and negative MSC membrane markers, were used. Positive markers used were CD105 FITC (R&D Systems); CD90 AL-647 (AbDSerotec,); HLA Class I FITC (Cytognos); CD73 PE (BD Bioscience) and CD166 PE (R&D Systems). Negative markers used were CD34 PE (BD Bioscience); HLA class IIPE (Cytognos); CD80 AL-647 (AbDSerotec); CD45 FITC (Cytognos) and CD31 FITC (Cytognos). Furthermore, suitable isotopic controls for FITC, PE (Cytognos) and AL-647 (AbDSerotec) were used as controls for specificity of the monoclonal antibodies.
The labelled cells were acquired with a flow cytometer FC500 MPL Cytomics (Beckman Coulter) using the MXP software (Beckman Coulter). Nonviable cells were discarded using the labeling reagent LIVE&DEAD (Invitrogen), and the collected data were analyzed with the CXP analysis software, version 2.1 (Beckman Coulter). Criteria for the administration of MSCs in our present clinical trial included a viability >95%, absence of microbial contamination (bacteria, fungus, virus or mycoplasma), expression of CD105, CD90, HLA I, CD73 and CD166 for >90% of cells and absence of CD34, CD80, HLA II, CD45 and CD31 (expression of each <5%), as assessed using flow cytometry (Figure 1).