22 μm syringe filter

The sugars were identified and separated with HPIC (Dionex ICS-5000, Thermo Fischer Scientific, CA, USA), with the same chemicals, separation method, analytical column, electrode and detector as used in [34 (link)]. Prior to the HPIC analysis, the samples were diluted 50 times and were filtered with 22 μm syringe-filter (Millipore, USA) into the HPIC vials. The sugars in the samples were quantified after comparing them to reference standards of rhamnose, galactose, glucose, xylose, fructose and glucuronic acid (chemicals for standards from Sigma-Aldrich, Israel).
The ethanol was measured with an ethanol assay kit (K-ETOH, Magazyme, Ireland) using a spectrophotometer (Tecan, infinite M200 PRO) at OD 340_nm.

FBS was centrifuged at 10,000 g overnight and then filtered through a 0.2 μm syringe filter (Millipore, Burlington, MA, USA). The exosome-deficient FBS was used for cell culture (DMEM supplemented with 10% exosome-free FBS). To isolate exosomes, the cell supernatant was collected and centrifuged at 2000 g and 10,000 g for 30 minutes at 4°C. The final supernatant was filtered through a 22 μm syringe filter (Millipore, Burlington, MA, USA) and centrifuged at 120,000 g for 70 minutes. Afterwards, the spheroidized vesicles were washed with PBS and centrifuged again at 120,000 g for 70 minutes. Finally, the extracellular markers were detected by Western blotting to validate the result.