Anti cd73 clone ad2

Flow cytometry analysis of stained hBM-MSC-EVs was performed with Apogee A50-Micro cytometer, unique high-resolution system dedicated for the reliable characterization of small particles. To ensure the specificity of obtained data, appropriate isotype controls were also included in gating strategy. hBM-MSC-EVs were stained with the following mouse monoclonal antibodies: FITC-conjugated anti-CD9 clone M-L13, anti-CD63 clone H5C6, and anti-CD81 clone JS-81 (all from BD Biosciences, San Jose, CA, USA) and PE-conjugated anti-CD44 clone BJ18, anti-CD73 clone AD2, and anti-CD90 clone 5E10 (all from BioLegend, San Diego, CA, USA) or appropriate isotype-match controls. Briefly, hBM-MSC-EVs suspended in 0.2 μm filtered PBS were incubated with antibodies for 30 minutes at 4°C. Prior to addition, all antibodies were centrifuged at 21,000× g for 20 minutes at 4°C to remove potential protein aggregates. Additionally, hBM-MSC-EVs were stained with PKH26 membrane dye at RT for 5 minutes in the dark and blocked with FBS, according to manufacturer’s instructions. The sample of PBS only with PKH26 and FBS was also prepared as a control. Stained EV samples were analyzed by Apogee A50-Micro flow cytometer (Apogee Flow Systems, Hemel Hempstead, UK), and the percentage of gated positive events was calculated by the Histogram software (Apogee Flow Systems).