Cd73 pe

To characterize MSCs derived from different sources (N-7 for each tissue of MSC isolation), the following conjugated antibodies were used: CD10 FITC, CD14 FITC, CD29 APC, CD44 FITC, CD45 PE-Cy7, CD73 PE, CD90 APC, CD105 AF488, CD146 PE, CD271 (NGFR) PE, (Exbio Antibodies, Vestec u Prahy, Czech Republic), VEGFR2, SSEA-4 PE (BioLegend, San Diego, CA, USA), CD133 PE, MSCA-1 APC (Miltenyi Biotec Inc., Bergisch Gladbach, Germany), CD34 PE-Cy5, HLA-ABC, HLA-DR (BD Bioscience, San Diego, CA, USA) and CD235a PE (Abcam, Cambridge, UK). As negative controls, mouse IgG1 and IgG2a conjugated with FITC, PE and PE-Cy5 (Dako Cytomation, Glostrup, Denmark, BD Bioscience, San Diego, CA, USA) were used. Briefly, cells from passage 3 were sampled in designated tubes of at least 250 000 cells in 100 µl, and incubated with the appropriate amount of antibodies according to the manufacturer’s recommendation. Cells were then washed with PBS, fixed in 4% paraformaldehyde overnight and at least 10 000 cells were recorded on BD FACSAria^TM Cell Sorter, BD LSR II (BD Bioscience, San Diego, CA, USA) or Apogee A-50 Micro flow cytometer (Apogee Flow Systems, Hertfordshire, UK). The obtained data were then analysed using FlowLogic™ Software (Inivai™ Technologies, Mentone, Australia).

Flow cytometry analysis was applied to distinct mesenchymal and hematopoietic stem cells. After three passages, cells were trypsinized and used for the experiment at a density of 10⁵ cells per ml of PBS. The trypsinized cells in PBS were then treated with 5 μl CD44-FITC (Exbio/Czech), CD90-FITC (Exbio/Czech), CD45-FITC (Exbio/Czech), CD73-PE (Exbio/Czech), CD34-PE (Exbio/Czech), and CD105-PE (Exbio/Czech) antibodies. In this study, Mouse IgG1-FITC, Mouse IgG1-PE, and Mouse IgG2a-PE were used as the controls in separate tubes. Cells were then incubated in a dark room for 30 min prior to being centrifuged in 1 ml of washing buffer and were then centrifuged at 1500 rpm for 5 min. The cells were finally resuspended for flow cytometry detection (BD FACS Calibur using BD bioscience USA, CA—San Jose), and the results were analyzed using FlowJo 7.6.1 software.