Particle concentration and size distribution of hiPSC-EVs were measured by NanoSight NS300 analyzer (Malvern Pananalytical, Malvern, UK) based on the nanoparticle tracking analysis (NTA). Atomic force microscopy (AFM) analysis of hiPSC-EV samples was performed by BioScope Catalyst AFM system (Brüker, Billerica, MA, USA), as previously described [13 (link), 15 (link)]. For flow cytometry analysis, hiPSC-EVs were stained with RNASelect dye (Thermo Fisher Scientific) and antibodies against CD9, CD34, CD45, CD63, CD81, CD90, CD105, Tra-1-60, KDR, lymphocyte function-associated 1 protein (LFA-1), and stage-specific embryonic antigen-4 (SSEA-4) and further analysis was performed with an Apogee A50-Micro flow cytometer (Apogee Flow Systems, Hemel Hempstead, UK). In addition, stained hiPSC-EVs were also imaged by ImageStreamX Mk II imaging flow cytometer, using 60× objective magnification and IDEAS Software (Luminex Corp., Austin, TX, USA.). Moreover, western blotting analysis was performed to compare relative levels of expression of CD9, CD63, syntenin, calnexin and β-actin in lysates obtained from hiPSC-EVs and their parental cells.
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