Thinwall polypropylene tube

Differential ultracentrifugation was performed as described previously by Théry et al. [23 ]. Cell-free urine samples (50 mL) were thawed at room temperature and vortexed before processing. Next, urine was centrifuged for 30 min at 2000 g (with braking) and 4°C in an Eppendorf 5810R benchtop centrifuge with A-4-62 swinging bucket rotor. The supernatant was transferred to a 38.5 mL Thinwall Polypropylene Tube (Beckman Coulter) and subjected to serial ultracentrifugation (acceleration: max; deceleration: max) at 4°C at 12,000 g (SW 32.1 Ti rotor with r_avg = 11.36 cm and adjusted k-factor = 2483.31, Beckman Coulter) and 110,000 g (SW 32.1 Ti rotor with r_avg = 11.36 cm and adjusted k-factor = 270.91, Beckman Coulter), for 45 min and 2 h, respectively. The crude pellet was resuspended in 16 mL PBS and passed through a 0.22 µm Whatman syringe filter (GE Healthcare life sciences) and EV were pelleted in a 17 mL Thinwall Polypropylene Tube (Beckman Coulter) by centrifugation at 110,000 g (acceleration: max; deceleration: max) and 4°C for 70 min. An additional washing step was performed by repeating the previous resuspension and pelleting step. The final pellet was re-suspended in 100 µL PBS and stored at −80°C until further use.

DTT treatment was performed as a modification of a previously described protocol [25 (link)]. Cell-free urine samples (50 mL) were thawed at room temperature and vortexed before processing. Urine was transferred to a 38.5 mL Thinwall Polypropylene Tube (Beckman Coulter) and centrifuged (acceleration: max; deceleration: max) at 4°C and 17,000 g (SW 32.1 Ti rotor with r_avg = 11.36 cm and adjusted k-factor = 1752.92, Beckman Coulter) for 10 min to pellet THP polymers. The supernatant was kept at 4°C, while the resulting 1.5 mL pellet was incubated with 1 mL of a freshly prepared dithiothreitol solution (50% DTT in aqua distilled) for 10 min at 37°C and vortexed to depolymerize THP (final DTT concentration: 200 mg/mL). The treated pellet and previous supernatant were mixed in a 38.5 mL Thinwall Polypropylene Tube (Beckman Coulter) and centrifuged again for 10 min at 17,000 g (acceleration: max; deceleration: max) and 4°C.
The resulting supernatant was used for uEV separation by BU ODG, as described above.

Isolation of EV was done from conditioned media after 48 hours of culturing GL261 in RPMI with 1% P/S and 5% EV-depleted FBS (see “Cell Culture”). The differential ultracentrifugation protocol consisted of subsequent centrifugation at 300×g for 10 min and 2000×g 10 min. Supernatants were filtered through 0.8μm filter (Sigma) and centrifuged for 100,000×g (k-factor of 220.1) 120 min in Quick-Seal® Polypropylene Tubes (Beckman) using Type 70 Ti in Optima XE ultracentrifuge (Beckman) to pellet EVs. To wash and concentrate EVs, pellets were resuspended in remaining supernatant supplemented with OptiMEM and concentrated by centrifugation at 100,000×g (k-factor of 190.7) for 120 min in Thinwall Polypropylene Tubes (Beckman) using MLS-50 Swinging-Bucket Rotor (Beckman) in an Optima Max-XP Ultracentrifuge. Final EV pellet was resuspended in DPBS and characterization of EVs was performed by size distribution analysis using nanoparticle-tracking analysis (NTA 3.2; Malvern), with screen gain set at 3.0 and camera level at 13.0.
Following procedures as described in intracranial tumor implantation method section EVs or an equal volume of carrier fluid (PBS) was injected intracranially. Microglia were isolated 16 and 40 hours after injection of EVs or DPBS following procedures, as previously described.