0.2 μm syringe fitted filters

In order to generate exosome-free media, exosomes present in FBS were removed by overnight centrifugation at 100,000× g followed by filtration through 0.2-μm syringe-fitted filters (Millipore, Burlington, MA, USA). This exosome-depleted FBS was used for cell culture (corresponding culture media supplemented with 10% exosome-free FBS). For exosome isolation, BICR-18 cell supernatants and plasma samples from AP-induced rats were collected and centrifuged at 2000× g and 10,000× g for 10 and 30 min, respectively, at 4 °C. The last supernatant was filtered through a 0.22-µm syringe filter (Millipore, Burlington, MA, USA) and ultra-centrifuged at 120,000× g for 70 min. After that, the pelleted vesicles were washed with PBS and centrifuged again at 120,000× g [16 (link)].
The quality of exosome preparation was verified by nanoparticle tracking analysis and by determining the presence of the exosomal marker TSG101 and the absence of calnexin by Western blot. The number of exosomes obtained was also quantified by measuring their protein content using a Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA).

In order to generate exosome-free medium, exosomes present in fetal bovine were removed by an overnight centrifugation at 100 000 g followed by filtration through 0.2-μm syringe-fitted filters (Millipore, Burlington, MA). This exosome-depleted fetal bovine serum was used for cell culture (DMEM supplemented with 10% exome-free fetal bovine serum). For the exosomes’ isolation, cell supernatants were collected and centrifuged at 2 000 xg and 10 000 xg for 10 and 30 min, respectively, at 4°C. The last supernatant was filtered through a 0.22 μm syringe filter (Millipore Burlington, MA) and ultracentrifuged at 120 000 xg for 70 min. After that, the pelleted vesicles were washed with phosphate-buffered saline (PBS) and centrifuged again at 120 000 xg for 70 min [20 (link)]. Quality of exosomes preparations was verified by nanoparticle tracking analysis, electron microscopy analysis of their size and shape and thus by determining the presence/absence of exosomal markers TSG101, and ALIX by Western Blot. The amount of exosomes obtained was also quantified by measuring their protein content using a Bradford assay, as previously described [20 (link)].