Gibco exosome depleted fbs

Cells were seeded in 10 cm diameter culture dishes at 500,000 cells/dish and maintained for 24 h in DMEM supplemented with 10 % Gibco™ exosome-depleted FBS (ThermoFisher Scientific, Waltham, MA). The medium was then replaced with fresh medium containing either 10 μM MOPIPP, synthesized as described [24 (link)], or 1 μM vacuolin-1 (Santa Cruz Biotechnology, Santa Cruz, CA) dissolved in DMSO. Control cultures contain an equivalent volume of the DMSO vehicle (0.1 %) in the medium. After 24 h, the medium was collected from the dishes (typically 10–12 dishes per condition) and the attached cells were pooled and counted with a Coulter Counter, model Z1 (Beckman-Coulter, Indianapolis, IN). Exosomes were isolated from medium using the Exo-spin™ Exosome Purification system (Cell Guidance Systems, St. Louis, MO). The medium was pre-cleared by centrifugation at 4° C, first at 300 × g for 10 min and then at 16,000 × g for 30 min. Then a volume of Buffer A equal to half the volume of medium was added and the mixture was incubated overnight at 4° C. The precipitate, enriched with exosomes, was collected by centrifugation at 16,000 × g for 1 h, and the pellet was re-suspended in 100 μl of Dulbecco’s phosphate-buffered saline (PBS), pH 7.4. The material was applied to an Exo-Spin column equilibrated with PBS, and the purified exosomes were eluted in 200 μl of PBS.

Cell culture supernatant (using cells cultured with media described above and 10% Gibco Exosome-Depleted FBS (ThermoFisher Scientific)) was centrifuged at 3400g for 15 min and the supernatant was collected. Extracellular vesicles were isolated from the supernatant using differential ultracentrifugation (Beckman Coulter Optima Max-XP Ultracentrifuge, rotor TLA120.2) as described in figure S1. Pellets were washed by resuspending in PBS and re-pelleting via a second round of ultracentrifugation. These pellets were then resuspended in PBS and incubated overnight at 4°C with magnetic CD9, CD63 and CD81 Dynabeads (Invitrogen). The exosomes-attached to the beads were magnetically separated from flow through using a magnetic microcolumns (μ columns from Miltenyi Biotec MACS). The flow through eluent (membrane particles and apoptotic bodies) was kept and re-pelleted for further analysis (2h at 245,000g). For Western blot and flow cytometry, the exosomes were not removed from the magnetic beads. For Nanoparticle Tracking Analysis or SEM imaging, exosomes were eluted from the beads using elution buffer (ExoFlow Exosome Elution Buffer, System Biosciences) as per manufacture’s protocol. This method was submitted to EV track.^{27 (link)}