Amicon ultra 2 centrifugal filters

For CCM and urine (40–60 ml), 10K molecular weight cut off (MWCO) Centricon^® Plus‐70 Centrifugal Filters (MilliporeSigma, Burlington, MA, USA) were used to concentrate the initial volume to 0.5 ml according to the manufacturer's instructions. qEV original 70 nm columns (IZON Science, Cambridge, MA) were used for EV separation. Briefly, the columns were brought to RT for 30 min and washed with PBS. Up to 0.5 ml of sample was loaded to the column. Fourteen sequential fractions of 0.5 ml were eluted by adding PBS. We identified EV enriched fractions (EVEF) as fractions 7 to 10 (Figure S2). EVEF were pooled and further concentrated using 10K MWCO Amicon^® Ultra‐2 Centrifugal Filters (MilliporeSigma) to a final volume of 100 μl.

EV original 70-nm columns (IZON Science, Cambridge, MA, USA) were used to separate the EVs as previously described[13 (link)]. Urine (40 mL) and plasma (2 mL) was concentrated to 0.5 mL using 10 kDa molecular weight cut off (MWCO) filters (Centricon^® Plus-70 Centrifugal Filters and 10 kDa MWCO Amicon^® Ultra-2 Centrifugal Filters, MilliporeSigma, Burlington, MA, USA). The columns were washed with phosphate-buffered saline (PBS). Samples (≤ 0.5 mL) were loaded onto the column. Fourteen sequential 0.5-mL fractions were eluted using PBS. The EV-enriched fractions (EVEF) were fractions 7–10. The EVEF were pooled and concentrated using the kDa centrifugal filters to a final volume of 100 μL.