Exo flow capture kit

To identify the surface markers of BMSCs, flow cytometry was carried out using the anti-CD34, anti-CD44, anti-CD45, anti-CD31, anti-CD105, anti-CD90, anti-HLA-DR, and anti-CD73 antibodies (abovementioned antibodies were all purchased from Thermo/eBio, information listed Supplemental Table 3) by Cytomics™ FC 500 (Beckman Coulter). The surface markers of EVs were assessed by flow cytometry analysis using a commercially Exo-Flow capture kit, including CD9, CD63, and CD81 flow antibodies (System Biosciences, CA, USA). The procedures were in line with published study [44 ]. In brief, isolated EVs were captured by microbead with CD9, CD63, and CD81 antibodies in the kit. Then, stained the Exo-microbead complexes with Exo-FITC, which features FITC bonded to a protein conjugated with the abovementioned EVs surface protein. Furthermore, the isotype control provided by those kits, which was negative to CD9, CD63, and CD81, was used as the control group for FACS. Finally, the EV complexes were evaluated and processed by FACS (Novocyte Flow Cytometer, ACEA Bioscience, Inc., MA, USA). In addition, data analysis was conducted using FlowJo software (Version 10).

Flow cytometry analysis (FACS) of EVs was performed using a commercially available Exo-Flow capture kit (System Biosciences, CA, USA) according to the manufacturer’s protocol. Briefly, isolated EVs were captured on microbead with CD9, CD63 and CD81 antibodies provided in the kit. The EV-microbead complexes were stained by Exo-FITC that features FITC conjugated to a protein which is bind EV surface protein. The EV complexes were analysed by FACS (Novocyte Flow Cytometer, ACEA Bioscience, Inc., MA, USA). Data acquisition and analysis were performed using NovoExpress software.