The serum used for the study was isolated from the blood of four healthy volunteers who provided written informed consent (permission of the local Bioethical Committee no. KB/430-16/13). Five mL of blood was collected into an anticoagulant-free tube (Becton Dickinson, Franklin Lakes, NJ, USA; 367955), incubated for 30 min at 20 °C then centrifuged at 1000 × g for 10 min at 4 °C. The supernatant (i.e., serum) was transferred to clean tubes and stored at −80 °C until use. Small extracellular vesicles were isolated by micro-SEC (size exclusion chromatography) from 0.5 mL of serum. Serum was pre-purified by a series of centrifugations at 1,000 and 10,000 × g for 10 and 30 min at 4 °C, respectively, then the supernatant was filtrated using a 0.22 µm syringe filter unit (Roth, Karlsruhe Germany; PA49.1). Filtered serum was loaded onto an Econo-Pac 10DG column (BioRad, Hercules, CA, USA; 732-2010) filled with 10 mL of Sepharose CL-2B (GE Healthcare, Chicago, USA; 17014001) at 6 cm length. The first 0.5 mL fraction was collected right after the sample had been loaded (void volume) then subsequent fractions (0.5 mL each) were eluted using PBS. The presence of EVs in the collected fractions was detected by Western blot using typical exosome markers (CD9, CD63, CD81, and TSG101 [2 (link),17 (link),18 (link),19 (link)]); EVs started to be eluted in fraction 7.
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