Pe conjugated mouse anti human cd73

For cell surface markers analyses, cells were treated with 0.5% (w/v) bovine serum albumin and double-stained using monoclonal antibodies, including phycoerythrin (PE)-conjugated mouse anti-human CD34 (BD Biosciences, Le-Pont-de-Claix, France) with fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD90 (Beckman Coulter, Villepinte, France), PE-conjugated mouse anti-human CD166 (Beckman Coulter, Villepinte, France) with FITC-conjugated mouse anti-human CD45 (Dako, Glostrup, Denmark), PE-conjugated mouse anti-human CD105 (Beckman Coulter, Villepinte, France) with FITC-conjugated mouse anti-human CD44 (Beckman Coulter, Villepinte, France), and PE-conjugated mouse anti-human CD73 (BD Biosciences, Le-Pont-de-Claix, France) with FITC-conjugated mouse anti-human leukocyte antigen-antigen D related (HLA-DR) (Beckman Coulter, Villepinte, France). Antibodies were used at a dilution of 1:10 (PE CD34, FITC CD90, PE CD166, FITC CD44, PE CD105, FITC HLA-DR and PE CD73) or 1:20 (FITC CD45). Appropriate isotype-matched control antibodies named FITC or PE mouse IgG1 (Dako, Glostrup, Denmark) were used in each analysis. Cells were then examined by flow cytometry using a BD LSR II flow cytometer (Becton Dickinson Biosciences, San Jose, California). Fluorescence intensity and percentage of antigen positive cells were determined for each surface marker.

Cells were harvested and blocked with 2% bovine serum albumin (BSA; Sigma-Aldrich, B2064) for 20 min at room temperature. Then, the cells were stained with fluorescein-conjugated antibodies for 40 min at room temperature in 1% BSA. After incubation, cells were washed 3 times and analyzed with MoFlo (Beckman, Brea, CA, USA) and associated software. The antibodies used for flow cytometry were as follows: PE-conjugated mouse anti-human CD29 (Biolegend, San Diego, CA, USA; 303004), PE-conjugated mouse anti-human CD73 (BD Biosciences, San Jose, CA, USA; 550257), PE-conjugated mouse anti-human CD90 (eBioscience, San Diego, CA, USA; 12-0909-42), PE-conjugated mouse anti-human CD105 (Biolegend, 323206), PE-conjugated mouse anti-human CD34 (BD Biosciences, 555822), PE-conjugated mouse anti-human CD45 (BD Biosciences, 560957), PE-conjugated mouse anti-human HLA⁻DR (BD Biosciences, 555561), and the PE-conjugated mouse IgG1 (BD Biosciences, 551436) as an isotype control.

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).