Sepharose cl 2b

EV were isolated as described in Peacock et al [12 (link)]. Briefly, NOF or eCAF were rinsed with PBS and incubated in serum-free DMEM for 24–72 h. Conditioned medium collected and centrifuged at 300 x g for 10 min, 2000 x g for 10 min and 10,000 x g for 30 min. The supernatant was reduced to 0.5 ml using a Vivaspin-20 (100 kDa molecular weight cut-off) column (GE Healthcare, Buckinghamshire, UK). EV were isolated by size exclusion chromatography using Sepharose CL-2B (GE Healthcare, Uppsala, Sweden) stacked in disposable Econo-Pac columns (Biorad, Watford, UK) and eluted in PBS. Where required, EVs were pelleted by ultracentrifugation at 100,000 x g for 1 h. Size profile and quantification of EV was performed by nanoparticle tracking analysis using a Zetaview instrument (Particle-Metrix) according to the manufacturer’s instructions.

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.

Peripheral blood was collected into a 5-milliliter BD Vacutainer Tube, incubated for 30 min at room temperature to allow clotting, and then centrifuged at 1000× g for 10 min to remove the clot. The serum was aliquoted and stored at −80 °C before further processing. Extracellular vesicles were isolated by micro-SEC (size-exclusion chromatography) from 0.84 mL of serum in each case. Serum was pre-purified by a series of centrifugations at 1000 and 10,000× g for 10 and 30 min at 4 °C, respectively; then, the supernatant was filtrated using a 0.22-micrometer syringe filter unit (Roth, Karlsruhe, Germany; PA49.1). Phosphate-buffered saline (PBS) was added to the filtered serum to equilibrate its final volume to 1 mL; then, the sample 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, IL, USA; 17014001) at 6 cm length. The column was left until dripping ceased (void volume); then, the first 1 mL fraction was eluted by loading 1 mL of PBS. Further fractions were eluted analogously; sEV were eluted in fractions 3 and 4 (F3 and F4). For further lipidomics analysis, 950 µL of fraction F3 was concentrated to 50 µL using Vivaspin500 ultrafiltration tubes (Sartorius, Göttingen, Germany; VS0102) according to the manufacturer's instructions.