Cd44 fitc

ASCs were further expanded in vitro and were analyzed at passages 4 to 5 by flow cytometry (FACSAria; BD Biosciences, Erembodegem, Belgium). Monoclonal antibodies against CD9‐PE, CD10‐PECy7, CD13‐PE, CD14‐PECy, CD19‐PECy7, CD29‐APC, CD49d‐PE, CD73‐PE, CD90‐APC, CD106‐PE‐Cy5, CD146‐PE, and CD166‐PE (BD Biosciences); CD45‐FITC (Miltenyi Biotech, Bergisch Gladbach, Germany); CD31‐FITC, CD34‐APC, CD44‐FITC, HLA‐ABC‐PE, and HLA‐DR‐PE (Immunotools GmbH, Friesoythe, Germany); and CD105‐PE (R&D Systems Inc., Minneapolis, Minnesota) were used. Analysis was performed on 10,000 cells per sample. The positive expression was defined as the level of fluorescence greater than 99% of the corresponding unstained cell sample.

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).