Exosome isolation

Exosomes were depleted from the complete media by ultracentrifugation for 70 min at 100,000 × g using an ultracentrifuge (Beckman Coulter) and SW28 swinging-bucket rotor. MDSCs (6×10⁶) were grown in a T175 flask for 72 h under normoxic conditions (5% CO₂ and 20% oxygen) at 37°C in a humidified incubator. The cell culture supernatant was centrifuged at 700 × g for 15 min to remove cell debris. To isolate exosomes, we employed a combination of two steps of the size-based method by passing through a 0.20-µm syringe filter and centrifugation with 100k membrane tube at 3,200 × g for 30 min followed by a single step of ultracentrifugation at 100,000 × g for 70 min [as described in our previous publication (16)].