Centricon filter unit

Cell-free urine samples were unfrozen overnight at 4 °C and centrifuged at 17,000 g for 10 min. The supernatant was kept and the 17,000 g pellet was treated with DTT (200 mg/mL, Sigma-Aldrich) for 10 min at 37 °C to release Tamm-Horsfall protein polymers, as previously described [31 ]. The DTT-treated pellet and the 17,000 g supernatant were mixed and centrifuged again at 17,000 g for 10 min. Then, supernatant was concentrated by ultrafiltration, using a 100 kDa cut-off Centricon filter unit (Millipore, Bedford, MA). One mL of the retained volume of concentrated urine was then loaded into a 10-mL sepharose CL-2B (Sigma) SEC column to isolate uEVs. For each sample, 20 fractions of 0.5 ml were collected [32 (link)].

The expression and purification of recombinant TTR (possessing or devoid of the C-terminal histidine tag), which was cloned into the pGEX-2T vector, was performed according to Ref. [34 (link)]. Briefly, the expression of TTR fused to glutathione-S-transferase (GST) in the E. coli BL21 pLys cell line was induced with 1 mM isopropyl β-d-1-thiogalactopyranoside. The cells were cultured for 3 h at 37 °C, harvested by centrifugation, washed and suspended in phosphate-buffered saline (PBS) containing 1 mM DTT and frozen at −80 °C. Prior to purification, the cell suspension was thawed and lysed by pipetting. DNase I (10 μg/mL), RNase A (10 μg/mL) and phenylmethylsulfonyl fluoride (0.2 mg/mL) were added during cell lysis, and the soluble fraction was obtained by centrifugation. TTR was purified using a two-step protocol. First, the supernatant (containing fusion protein) was loaded on a glutathione-Sepharose column equilibrated with PBS, and contaminating proteins were washed out with PBS. Then, TTR was digested from the resin with thrombin (Calbiochem) at room temperature for up to 24 h. TTR-containing fractions were collected by washing the column with PBS, concentrated using a Centricon filter unit (Millipore), loaded onto a Superdex 200 column (30/300 GL, GE Healthcare) equilibrated with Tris buffer and separated using Akta Explorer (Amersham Biosciences).