The concentration of EVs was estimated based on the total protein content, determined using a bicinchoninic acid (BCA) protein assay kit (Cat. No. E-BC-K318-M, Elabscience). Nanoparticle tracking analysis (NTA) with a ZetaView PMX 110 (Particle Metrix) was used to analyze the distribution and size of EVs, while Hitachi H-7650 transmission electron microscope (TEM; Hitachi) was used to identify the morphologies of EVs. To assess the effectiveness and stability of each batch of EVs, cellular functional tests and NTA analysis were employed. Flow cytometry on a FACSCANTO II (BD Biosciences) was employed to resolve EV surface marker proteins, including CD63, CD81, and TSG101, as previously described [48 (link), 49 (link)], and FlowJo software (Tree Star Inc) was used to analyze the results. The CD63 polyclonal antibody (Cat. No. 25682-1-AP, Proteintech), CD81 monoclonal antibody (Cat. No. SC7637, Santa Cruz Biotechnology), TSG101 polyclonal antibody (Cat. No. 14497-1-AP, Proteintech) were used as primary antibodies. Alexa Fluor 488-conjugated goat anti-rabbit IgG (Cat. No. 111-545-144, Jackson ImmunoResearch) and Alexa Fluor 594-conjugated donkey anti-mouse IgG (Cat. No. 715-585-151, Jackson ImmunoResearch) were used as secondary antibodies.
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