Immunophenotyping of hHFMSCs by Flow Cytometry

Immunophenotyping of hHFMSCs was carried out using a BD FACSCalibur Cell Sorting System (BD Calibur) as previously described [25 (link)] with minor modifications. hHFMSCs were treated with TrypLE and stained with monoclonal antibodies anti-CD44, anti-CD34, and anti-CD166 (1 : 100, BD) in addition to antibodies used in our previous study [25 (link)]. hHFMSC^OCT4 and floating cells were treated with TrypLE. Live cells were identified by 7-amino actinomycin (7AAD) exclusion and analyzed for EGFP expression. To detect the expression of hematopoietic markers, single cells were stained with fluorochrome-conjugated monoclonal antibodies PE-anti-CD45 (1 : 100, BD Pharmingen) and PE-Cy5-anti-CD34 (1 : 100, Cell Signaling Technology). For CD133 detection, Alexa Fluor-555 goat anti-mouse IgG (1 : 200, Cell Signaling Technology) was used as the secondary antibody.