Stericuprvp

Isolation, purification, and characterization of EVs were performed by following MISEV 2018 Guidelines (19 (link)). Briefly, EDTA-blood was centrifuged 1,200 × g for 15 min at room temperature to obtain platelet-free blood plasma. Plasma was further centrifuged at 1,000, 2,000, and 3,000 × g for 15 min at 4°C, discarding the pellet to clean the cell debris. To prepare EV pellet for Nanosight and Flow Cytometry, 1.5 mL of fresh plasma was transferred into an ultracentrifuge tube (Quick-Seal^®-Round-Top, Polypropylene, 13.5 mL-Beckman Coulter, Inc.) and filled up with PBS, filtered with 0.10 μm pore size membrane (StericupRVP, 0.10 μm, polyethersulfone filter- Merck Millipore) to minimalize the background contribution of interfering particles. Plasma was then ultracentrifuged (BeckmanCoulter Optima-MAX-XP) at 110,000 x g for 75 min at 4°C, to obtain an extracellular vesicles-rich pellet. The pellet was re-suspended with 500 μL triple 0.10 μm pore size membrane-filtered PBS. To prepare the EV pellet for miRNA extraction, 1.5 mL of fresh plasma was transferred into an ultracentrifuge tube (Centrifuge bottles polycarbonate, 10.4 mL-Beckman Coulter) and filled up with PBS. Plasma was then ultracentrifuged (BeckmanCoulter Optima-MAX-XP) at 110,000 x g for 75 min at 4°C, decanted, and the EV pellet was kept at −80°C until miRNA extraction.

Blood samples were collected in ethylenediamine tetra-acetic acid (EDTA) tubes and processed within 2 h from the phlebotomy as described below. For each subject, an aliquot of 3 mL of synovial liquid was centrifuged at 1000, 2000, and 3000× g for 15 min at 4 °C. After each centrifugation step, pellets were discarded to remove cell debris. The resulting supernatants were subjected to ultracentrifugation (UC) at 110,000× g for 75 min at 4 °C in polypropylene UC tubes (Beckman Coulter; Brea, CA, USA) filled with phosphate-buffered saline (PBS) previously filtered through a 0.10 μm pore-size polyethersulfone filter (StericupRVP, Merck Millipore; Burlington, MA, USA). The EV-rich pellet obtained by UC was then resuspended in 0.5 mL of triple-filtered PBS (pore size 0.1 µm) to perform NTA and flow cytometry analysis.
Experiments carried out using either the soluble component or the membrane pellet were preceded by disrupture of intact EVs by multiple freeze and thaw cycles: broken EVs were repelleted at 100,000× g for 60 min [40 (link)] and the membrane pellet or luminal content obtained were then resuspended in filtered PBS.