MSC phenotype

The expression of cell surface protein was investigated by flow cytometry technique. The MSCs were trypsinized and incubated with specific PE- or fluorescein isothiocyanate-conjugated (FITC)-conjugated anti-CD31, CD45, CD90, and CD105 fluorescent dye conjugated anti-human antibodies for 30 min at 4 °C in a dark place. Afterwards, the cells were washed and re-suspended in PBS. The suspended cells were applied in FACS caliber (BD Biosciences) for cell surface protein expression measurement. Flowcytometric data was analyzed using FlowJo software (FlowJo, LLC). Positive and negative controls were also evaluated in each run. Furthermore, to confirm the mesenchymal phenotype of isolated cells, these cells were differentiated into osteogenic and adipogenic lineages as previously described [28]. The cells were sub-cultured and passage-3 MSCs were used for all experiments.