After 48 h incubation, the conditioned media was collected and centrifuged at 300xg for 10 min to pellet cells. The supernatant was collected and spun at 2,000xg for 20 min to remove dead cells, followed by storage at -80 °C until EV isolation.
To isolate the EVs, the conditioned medium underwent differential ultracentrifugation using a 30% sucrose/deuterium oxide (Sigma-Aldrich, Poole, UK) cushion, made up to a density of 1.210 g/cm3 [22 ]. The conditioned media was passed through a 0.22 µm filter (Sarstedt, Leicester, UK), loaded onto a 30% sucrose cushion, and then centrifuged at 100,000xg for 1 h 45 min on an SW28Ti rotor, 25PC Polycarbonate open-top tubes (Scientific Laboratory Supplies, Nottingham, UK) and using an L8-M Ultracentrifuge; k-factor 296.8 (Beckman Coulter, High Wycombe, UK). Using a 21G needle and syringe, 3-4 ml of EV suspension was collected at the interface between the sucrose cushion and conditioned media. DPBS was added to this suspension, and it was centrifuged on a Type 70.1Ti fixed angle rotor at 100,000xg for 60 min to pellet pure EVs. All ultracentrifugation experiments were performed at 4 °C and EV pellets were stored at -80 °C. For clarity, this paper uses the term ‘extracellular vesicles’ to describe both exosomes and microvesicles which are smaller than 0.22 µm and float at a density of < 1.2 g/ml, and hence represent small EVs.
To isolate the EVs, the conditioned medium underwent differential ultracentrifugation using a 30% sucrose/deuterium oxide (Sigma-Aldrich, Poole, UK) cushion, made up to a density of 1.210 g/cm3 [22 ]. The conditioned media was passed through a 0.22 µm filter (Sarstedt, Leicester, UK), loaded onto a 30% sucrose cushion, and then centrifuged at 100,000xg for 1 h 45 min on an SW28Ti rotor, 25PC Polycarbonate open-top tubes (Scientific Laboratory Supplies, Nottingham, UK) and using an L8-M Ultracentrifuge; k-factor 296.8 (Beckman Coulter, High Wycombe, UK). Using a 21G needle and syringe, 3-4 ml of EV suspension was collected at the interface between the sucrose cushion and conditioned media. DPBS was added to this suspension, and it was centrifuged on a Type 70.1Ti fixed angle rotor at 100,000xg for 60 min to pellet pure EVs. All ultracentrifugation experiments were performed at 4 °C and EV pellets were stored at -80 °C. For clarity, this paper uses the term ‘extracellular vesicles’ to describe both exosomes and microvesicles which are smaller than 0.22 µm and float at a density of < 1.2 g/ml, and hence represent small EVs.