Cd146 pe

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.

LEV samples were analyzed by high-sensitivity flow cytometry using a standardized Gallios flow cytometer (Beckman Coulter, Villepinte, France) as previously described [28 (link)]. Briefly, a 1:500-diluted 30 µL sample was incubated for 15 min with an appropriate amount of specific antibody and 10 µL of Annexin V-FITC (AnnV-FITC, Tau Technologies, Kattendijke, The Netherlands). Binding buffer solution (400 µL, as described in Section 2.2.1) was then added to improve the binding of AnnV to phosphatidylserines. LEV count beads (30 µL, Biocytex, Marseille, France) were added to determine the concentration of LEVs in each sample. The flow cytometer settings and LEV gating were performed with Megamix beads (Biocytex, Marseille, France). Positive-AnnV-LEVs were defined as total of LEVs. Specific fluorescent antibodies (ICAM-1-directed CD54-PE, #IM1239V; CD31-PE, #PN-IM2409 and CD146-PE, #AD7483; Beckman Coulter, Villepinte, France) were used to characterize LEV surface antigens. Analysis was performed with Kaluza Analysis software 1.2 (Beckman Coulter, Villepinte, France), as previously described [29 (link)]. A total 10⁸ LEV samples were stored at −80 °C until further use.

Flow cytometry analysis was performed on a Guava easyCyte 6HT 2 L flow cytometer (Merck Millipore, Darmstadt, Germany) as described before [29 (link)]. Before measurement, 40,000 cells were resuspended in 195 μl 1× PBS and incubated after addition of 5 μl CD34-PE, CD56-PE, CD146-PE, IgG1-PE, IgG1-FITC, CD90-PE, CD105-PE, HLA-DR-PE, CD45-PE (all from Beckman Coulter, CA, USA), CD49a-FITC (MiltenyiBiotec GmbH, Bergisch Gladbach, Germany), CD14-PE (Thermo Scientific, MA, USA), CD19-PE (BioLegend, CA, USA), or CD73-PE (Becton Dickinson, NJ, USA) for 30 min at 4 °C in the dark following a washing step with 1× PBS. Histograms and dot plots were generated with a minimum of 5000 events at a sample flow rate of 1.8 μl/ml. Positive staining was obtained by comparison with isotype control set as 99% negative or comparison to control (negative) cells.