Mesenchymal and Hematopoietic Cell Markers Analysis

Evaluation of the expression of mesenchymal stem cell surface markers (CD73 and CD44), embryonic stem cell surface marker (OCT-4), and hematopoietic cell marker (CD45) was done by flow cytometric analysis as described previously [28 (link)]. Cells (10⁵ cells/100 μl) were gently washed in PBS containing 2% FBS and incubated separately with PE-conjugated mouse anti-human CD73 (561014; BD Pharmingen), CD44 (550989; BD Pharmingen), and CD45 (560975; BD Pharmingen) in darkness for at least 40 min at 4 °C. Evaluation of OCT-4 expression was analysed using indirect intracellular flow cytometry. The cells were permeabilized with 0.1% saponin. Then primary rabbit antihuman OCT-4 antibody (ab19857, Abcam) was added for 40 min and then incubated with FITC-conjugated goat anti-rabbit Ig (Sigma-Aldrich) for 30 min. As negative controls, isotype IgG (555748; BD Pharmingen) was used. Afterward, cells were washed twice with PBS-FBS, fixed in 1% formaldehyde solution, and analyzed by a flow cytometer (Partec GmbH).