Cd73 pe

The International Society for Cell Therapy previously suggested the following minimal criteria to define human MSCs [18] (link): expression of CD105, CD73, and CD90, and no expression of CD45, CD34, CD14, CD11b, CD79α, CD19, or HLA-DR surface molecules. Cells were harvested by treatment with 0.05% trypsin-EDTA (Sigma-Aldrich, St Louis, MO) for 3 min at 37°C, recovered by centrifugation at 400× g for 5 min, washed twice in ice-cold PBS containing 2% FBS, and re-suspended at 1×10⁵ cells/antibody test. The expression of specific MSC markers was assessed using the following antibodies: CD14-FITC, CD31-PE, CD105-FITC, CD34-PE, CD45-PE, CD73-PE, and CD90-APC (all purchased from Abcam, Cambridge, UK). Negative control staining was performed using FITC/PE/APC-conjugated mouse IgG₁ isotype antibodies (Abcam). After incubation for 30 min at room temperature in the dark, the cells were washed with PBS, resuspended in 100 µL PBS, and analyzed by a MACSQuant flow cytometer (Miltenyi Biotec, Gladbach, Germany).

Phenotyping of cell surface markers was performed by flow cytometry. The cells were stained with CD34-PE and CD45-FITC (all from BD Biosciences, USA), CD90-FITC (Dako, USA), CD73 PE (Abcam, USA) and isotype controls IgG1-FITC (Dako, USA), IgG1-PE (BD Biosciences, USA), and IgG2A-APC (BD Biosciences, USA). Flow cytometry data were acquired using a Guava EasyCyte 8HT flow cytometer and analysed using ExpressPro software (Merck Millipore, USA) comparing unlabelled, marker-labelled and isotype control populations in FL-1, FL-2 and FL-4 channels.