Mouse anti human cd44

The hAF cells (3 samples) were used to determine MSCs marker expression. The hAF cells were trypsinized with 0.25% trypsin-EDTA and centrifuged at 2,035 g for 6 min at room temperature and they were then incubated with monoclonal antibodies; fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD34, CD90 (Biolegend, San Diego, USA), mouse anti-human HLA-ABC (Immuno Tools GmbH, Friesoythe, Germany) and mouse anti-human fibroblast (EDM, Millipore Crop, UK), as well as phycoerythrin (PE)-conjugated mouse anti-human CD31, CD117, HLA-DR (Immuno Tools GmbH, Friesoythe, Germany), mouse anti-human CD44 (Pierce Biotechnology, Rockford, USA), mouse anti-CD45 (Biolegend, San Diego, USA) and mouse anti-human CD73 (Life Technologies, California, USA) for 1 h at 4 °C. FITC mouse isotype control and PE mouse isotype control (Biolegend, San Diego, USA) were used as negative controls. Cell fluorescence was evaluated using FACscan (Becton Dickinson, Lincon Park, NJ) and analyzed using the CellQuest Pro 9.0 software (Becton Dickinson). The data were presented as mean ± SEM.

The MSCs population in the hAF cell samples was examined by observing the expression of MSC specific cell surface proteins, 3 hAF cell samples with duplicate were incubated with monoclonal antibodies; phycoerythrin (PE)-conjugated mouse anti-human CD31, CD117, HLA-DR (Immuno Tools GmbH, Friesoythe, Germany), mouse anti-human CD44 (Pierce Biotechnology, Rockford, USA), mouse anti-CD45 (Biolegend, San Diego, USA) and mouse anti-human CD73 (Life Technologies, California, USA), as well as fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD34, CD90 (Biolegend, San Diego, USA) and mouse anti-human HLA-ABC (Immuno Tools GmbH, Friesoythe, Germany). PE mouse isotype control and FITC mouse isotype control (Biolegend, San Diego, USA) were used as negative controls. Cell fluorescence was evaluated using FACscan (Becton Dickinson, Lincon Park, NJ) and analyzed using CellQuest Pro 9.0 software (Becton Dickinson).