Cd34 ecd

Characterization of the SVF cell subpopulations was performed according to the recommendations of the International Federation of Adipose Therapeutics and Sciences (IFATS) and the International Society for Cellular Therapy (ISCT). Briefly, SVF suspension was digested with DNase I 10U/mL (Roche Diagnostics, Indianapolis, Indiana, USA) in Dulbecco’s Phosphate Buffered Serum (DPBS) Ca⁺⁺/Mg⁺⁺ free medium containing 0.1 mM ethylenediaminetetraacetic acid, 25 mM Hepes, 1% fetal calf serum for 15 minutes at 37°C and filtered through a 70-μm nylon cell strainer to eliminate the majority of cell aggregates. Cells were centrifuged, resuspended, and labeled 20 minutes at 4°C with the following fluorochrome-conjugated antibodies: CD14-FITC, CD90-FITC, CD146-PE, CD34-ECD, CD45-PC5 (Beckman Coulter, Miami, FL, USA) or their isotype control to determine nonspecific fluorescence. Red blood cells were lysed in NH₄Cl for 10 minutes at 4°C before cells were centrifuged and resuspended in DPBS Ca⁺⁺/Mg⁺⁺. NucBlue (Thermo Fisher Scientific, Waltham, MA, USA), allowing discrimination of viable and dead cells, was added for 5 minutes before flow cytometry analysis on a NAVIOS flow cytometer (Beckman Coulter). CD45 negative cells were discriminated in CD34⁻CD146⁺ and CD34⁺CD146⁻CD90⁺ described as regenerative perivascular cells, and CD34⁺CD146⁺ as endothelial cells.

Immune-phenotypical characterization was performed by FACS analysis on immune-selected hDPSCs at passage 1. The expression of the typical mesenchymal stem cells (MSCs) markers, i.e., CD73, CD90, CD105, CD34, CD45, HLA-DR, was evaluated, as previously described by Pisciotta et al. [27 (link)]. Cells were stained with the following fluorochrome-conjugated antibodies (Abs): anti-human-CD73-PE-CY7, -CD90-FITC, -CD105-APC, -CD45-PE and -HLADR-PE-CY7 (all from BD Biosciences, Franklin Lakes, NJ, USA), and -CD34-ECD (Beckman Coulter, Fullerton, CA, USA). A minimum of 10,000 cells per sample was acquired and analyzed by using the Attune Acoustic Focusing Flow Cytometer (Attune NxT, Thermo Fisher, Waltham, MA, USA). Data was analyzed by FlowJo 9.5.7 (Treestar, Inc., Ashland, OR, USA) under MacOS 10.