P70at rotor

The cells were cultured with DMEM supplemented with 10% (v/v) sEV-depleted FBS and 1% (v/v) penicillin and 1% (v/v) streptomycin (all from Life Technologies, Carlsbad, CA, USA). Cells were cultured to 80% confluence, and the supernatants were collected after 72 h post-treatment. For the dissection of conditioned medium (CM), whole CM (10 mL/10 cm dish) was collected and centrifuged at 2000× g for 10 min to eliminate cell debris. One half (5 mL) was used as whole CM, concentrated, and used to treat cells, and the other half (5 mL) was further processed.
The CM was further processed using a sterile 0.22-µm filter (GE Healthcare Life Sciences, Little Chalfont, UK) and they were transferred into new ultracentrifugation tubes (Hitachi, Chiyoda, Tokyo, Japan) and centrifuged at 100,000× g for 2 h at 4 °C in a Hitachi CP100NX (Hitachi, Chiyoda, Tokyo, Japan) ultracentrifuge with a P70AT rotor (Hitachi, Chiyoda, Tokyo, Japan).
The last supernatants containing sEV-depleted FBS were collected as denominated supernatant fraction (SN), and the pellets (sEV fraction) were resuspended at 200 µL PBS (MP Biomedicals, Illkrich-Graffenstaden, France) or medium depending on if they were used for characterization or functional assays.
All relevant data of our experiments were submitted to the EV-TRACK knowledgebase (EV-TRACK ID: EV220413) [13 (link)].

The exosomes were isolated from the plasma samples by ultracentrifugation. Briefly, approximate 4 ml plasma was thawed on ice and centrifuged at 3000 × g for 10 min to remove possible cell residues. After being filtered with 0.22 μm filter membranes, blood samples were balanced by adding phosphate buffer saline (PBS). Cleared blood samples were centrifuged at 120,000 × g for 10 h using P70AT rotor (Hitachi, Japan). All centrifugal steps were kept at 4 °C. Exosome fractions were resuspended in 250 μL PBS for the following RNA extraction.

EV was isolated from serum samples using ultracentrifugation (UC). Briefly, 500 μL of serum was centrifuged at 2500 × g for 10 min (4°C) followed by another centrifugation at 10000 × g for 30 min to pellet cell debris. The supernatant was then filtered through a 0.22-μm cellulose acetate centrifuge filter (Costar, USA), and the filtrate was diluted with PBS into a final volume of 5 mL. Crude EVs were pelleted by ultracentrifugation at 110,000 × g (P70AT rotor, Hitachi, Japan) for 5 h. Afterwards, the pellets were resuspended with PBS and ultracentrifuge at 110,000 × g for 70 min. The final EV pellets were resuspended with 50 μL of PBS and stored at -80°C for further analysis.