Medium fractionation and EV preparation

Centrifuge the conditioned medium at 1,500 x g for 0h 20m 0s at 4°C in a Sorvall RC 6+ centrifuge with fixed angle rotor F14S-6X250y FiberLite

Pour the supernatant into a new container without disturbing the pellet.

Centrifuge the supernatant at 10,000 x g for 0h 30m 0s at 4°C in a Sorvall RC 6+ centrifuge with fixed angle rotor F14S-6X250y FiberLite

The supernatant fractions at each step were collected and treated with methanol/chloroform to precipitate proteins which were then collected by centrifugation

Pellet fractions were resuspended in sample buffer to achieve a 20-fold concentration.

The sedimented fractions at each step were also collected and resuspended in sample buffer.

All the fractions were analyzed by immunoblot.

Centrifuge the conditioned medium at 1,500 x g for 0h 20m 0s at 4°C in a Sorvall RC 6+ centrifuge with fixed angle rotor F14S-6X250y FiberLite

Pour the supernatant into a new container without disturbing the pellet.

Centrifuge the supernatant at 10,000 x g for 0h 30m 0s at 4°C in a Sorvall RC 6+ centrifuge with fixed angle rotor F14S-6X250y FiberLite

Pour the supernatant into a new container without disturbing the pellet.

Add 35 ml of the collected supernatant into a single 38.5 ml ultra-clear tube. Repeat this step 5

times to fill a total of six 38.5 ml ultra-clear tubes.

Centrifuge at ~100,000 x g (29,500 RPM) for 1h 30m 0s using a SW32 Ti rotor at 4°C at maximum acceleration and brake.

Aspirate the supernatant, put the tubes upside down on paper towel to dry any residue of

medium. Aspirate the residue by vacuum system if needed.

Resuspend the pellet by adding 500 uL of PBS (pH 7.4) buffer to the bottom of each tube, put the

tubes on a compatible rack, and rigorously shake the tubes on orbital shaker in cold room for0h 30m 0s .

Combine the resuspend pellet (~500 uL x 6 tubes = 3mL; +1mL PBS) and centrifuge again at 120,000 x g (36,500 RPM) x 1h 0m 0s at 4°C in SW-55 rotor

Resuspend the pellet again in 200uL PBS Buffer and add 1mL of 60% (1.8M) sucrose buffer (20mM Tris 7.4, 150 mM NaCl) and vortex to mix the sample evenly. The final sucrose concentration should be above 50% measured by refractometer.

Aliquots of 40% (5 ml) and 10% (2 ml) sucrose buffer were sequentially overlaid above the sample. The tubes were then centrifuged at 150,000×g for 16h 0m 0s at 4°C in an SW41 Ti swinging-bucket rotor (Beckman Coulter)

After centrifugation, 0.5 ml fractions were collected from top to bottom and samples were analyzed by SDS-PAGE and immunoblot.