Cellquest pro 9

Cell surface antigens were used to characterize the MSC properties by flow cytometry. The cells were detached with 0.25% trypsin-EDTA and centrifuged at 3,700 rpm for 6 min at room temperature to obtain the cell pellets. Non-specific binding was then blocked with 10% human AB serum at 4 °C for 30 min. For the detection of Oct-4, the cells were permeablised with 1% Triton X-100 (amresco®, Ohio, USA) for 1 min before incubated with antibody. Detroit 551 (fibroblast) (Sigma-Aldrich, USA) was used a negative control for the detection of Oct-4. Subsequently, cells pellets were incubated with the following monoclonal antibodies: Mouse anti-human CD31-PE (Immuno Tools GmbH, Germany), CD34-FITC (Immuno Tools GmbH, Germany), CD44-PE (Pierce Biotechnology, USA), CD45-PE (Immuno Tools GmbH, Germany), CD73-PE (Immuno Tools GmbH, Germany), CD90-FITC (Biolegend, USA), CD105-PE (Pierce Biotechnology, USA), HLA-ABC-FITC (Immuno Tools GmbH, Germany), HLA-DR-PE (Immuno Tools GmbH, Germany), and Oct-4-Alexa Fluor® 488 conjugate (Sigma-Aldrich, USA). Isotype antibodies served as a control to exclude nonspecific binding. Quantitative analysis was performed using FACScan (BD Biosciences) and the results were analyzed using CellQuest™ Pro 9.0 software (BD Biosciences).

To characterize hAF-MSCs, the cell surface markers of cells cultured in basic media containing 10% FBS or 10% hPL were evaluated. The cells were trypsinized with 0.25% trypsin-EDTA at 37°C for 1 min and centrifuged at 2,035 × g for 6 min at room temperature to obtain the cell pellets. Then, non-specific binding was blocked using 10% human AB serum [serum from type AB donors; processed by our laboratory and inactivated at 53°C for 90 min as previously described (16 (link))] at 4°C for 30 min. Subsequently, they were incubated with the following monoclonal antibodies: Mouse anti-human CD34-FITC (cat. no. 343504; BioLegend, Inc.), CD44-FITC (cat. no. 21810443; ImmunoTools GmbH), CD45-phycoerythrin (PE; cat. no. 304008; BioLegend, Inc.), CD73-PE (cat. no. 21270734; ImmunoTools GmbH), HLA-ABC-FITC (cat. no. 21159033; ImmunoTools GmbH) and HLA-DR-PE (cat. no. 21388994; ImmunoTools GmbH). Cell surface marker expression was detected using a FACScan (BD Biosciences) and analyzed using CellQuest™ Pro 9.0 software (BD Biosciences).