Sev depleted fbs

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. Supernatants were centrifuged at 2000× g for 10 min at 4 °C and filtered using a sterile 0.22-μm filter (GE Healthcare Life Sciences, Little Chalfont, UK) to eliminate debris, and they were transferred into new ultracentrifugation tubes (Beckman Coulter, Mississauga, ON, Canada) and centrifuged at 100,000× g for 2 h at 4 °C in an Optimal-90K ultracentrifuge with a 60 Ti rotor (Beckman Coulter, Mississauga, ON, Canada). The last supernatants containing exosome-depleted FBS were removed, and the pellets were resuspended in 200-μL PBS (MP Biomedicals, Illkrich-Graffenstaden, France).

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)].