Anti cd73 pe

Both hASCs and DFAT, at passage 0, were suspended in a phosphate buffer solution (PBS) (Sigma-Aldrich) with 5% human serum (Catalog Number S4200, Biowest, Nuaill, France) at a density of 10 × 10⁵ per tube. The cells were immunostained with anti-human primary antibodies (mAbs) conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) for 20 min at 4 °C in the dark. The following mAbs were used: anti-CD13 PE, anti-CD14 FITC, anti-CD34 FITC, anti-CD45 PE, anti-CD73 PE, anti-CD90 PE, anti-CD105 PE (Beckman Coulter, Milan, Italy). After washing in PBS with 5% human serum, the cells were centrifuged at 1300 rpm for 1 min. The cells were analyzed with a Navios flow cytometer (Beckman Coulter). Data were acquired, displayed and elaborated by Kaluza 1.2 software package (Beckman Coulter) and the positive cells were counted and compared with the signal of corresponding immunoglobulin isotypes.

To characterize MSCs that were used for EV production, 100 µL of cell suspension (1 × 10⁶/mL) was stained with anti-CD73 PE (0.3 µg/mL) and anti-CD90 APC AF750 (1 µg/mL; all from Beckman Coulter) to detect the MSC surface markers ecto-5′-nucleotidase and Thy-1, respectively. Staining was performed for 15 min in the dark. Stained samples were diluted fivefold in PBS and analyzed on a CytoFlex-LX flow cytometer (Beckman Coulter) equipped with 375 nm, 405 nm, 488 nm, 561 nm, and 638 nm lasers. Data were acquired for 3 min at a flow rate of 30 μL/min and analyzed using Kaluza 2.1 (Beckman Coulter).

To characterize MSC-derived EVs, EV suspensions isolated as described above were diluted 1:100 in 0.1 µm filtered AnnexinV (Anx5) binding buffer (ABB) and stained with APC-conjugated Anx5 (0.1 µg/mL; Becton Dickinson) as marker for phosphatidylserine (PS) as well as with anti-CD73 PE (0.6 µg/mL) and anti-CD90 APC-AF750 (2.5 µg/mL; both from Beckman Coulter). To remove any precipitates, fluorochrome conjugates were centrifuged at 18,000 × g for 10 min at 4 °C before use. Stained samples were diluted fivefold in ABB and analyzed on a CytoFlex-LX flow cytometer. Fluorescent-green silica particles (1.0 µm, 0.5 µm, 0.1 µm; excitation/emission 485/510 nm; Kisker Biotech, Steinfurt, Germany) were used for calibration, the triggering signal was set to violet side scatter (405 nm), and the EV gate was set below the 1 µm bead cloud as previously described (Tripisciano et al. 2017 (link); Weiss et al. 2018 (link)). Data were acquired for 2 min at a flow rate of 10 μL/min and analyzed using Kaluza 2.1 (Beckman Coulter). To calculate the number of events for each sample, the dilution factor during sample preparation and staining was considered. MSC-EVs were defined as Anx5⁺ events in the EV gate.