TITLE: Medium fractionation and EV preparation
AUTHORS: wusj, Nancy C. Hernandez Villegas, schekman

[DESCRIPTION]
This protocol describes the characterization of the extracellular alpha-synuclein and DNAJC5

[STEPS]
SECTION: Extracellular vesicle (EV) preparation
8. Centrifuge the conditioned medium at 1,500 x g for 20 min at 4 °C in a Sorvall RC 6+ centrifuge with fixed angle rotor F14S-6X250y FiberLite  

SECTION: Extracellular vesicle (EV) preparation
9. Pour the supernatant into a new container without disturbing the pellet.
SECTION: Extracellular vesicle (EV) preparation
10. Centrifuge the supernatant at 10,000 x g for 30 min at 4 °C in a Sorvall RC 6+ centrifuge with fixed angle rotor F14S-6X250y FiberLite
SECTION: Extracellular vesicle (EV) preparation
11. Pour the supernatant into a new container without disturbing the pellet.
12. 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.
SECTION: Medium fractionation
1. Centrifuge the conditioned medium at 1,500 x g for 20 min at 4 °C in a Sorvall RC 6+ centrifuge with fixed angle rotor F14S-6X250y FiberLite  
SECTION: Medium fractionation
2. Pour the supernatant into a new container without disturbing the pellet.
SECTION: Medium fractionation
3. Centrifuge the supernatant at 10,000 x g for 30 min at 4 °C in a Sorvall RC 6+ centrifuge with fixed angle rotor F14S-6X250y FiberLite

13. Centrifuge at ~100,000 x g (29,500 RPM) for 90 min using a SW32 Ti rotor at 4 °C at maximum acceleration and brake.
14. 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.



15. 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 for 30 min.



16. Combine the resuspend pellet (~500 uL x 6 tubes = 3mL; +1mL PBS) and centrifuge again at 120,000  x g (36,500 RPM) x 60 min at 4 °C in SW-55 rotor



17. 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.



18. 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 960 min at 4 °C in an SW41 Ti swinging-bucket rotor (Beckman Coulter)




19. After centrifugation, 0.5 ml fractions were collected from top to bottom and samples were analyzed by SDS-PAGE and immunoblot.
SECTION: Medium fractionation
4. The supernatant fractions at each step were collected and treated with methanol/chloroform to precipitate proteins which were then collected by centrifugation
SECTION: Medium fractionation
5. Pellet fractions were resuspended in sample buffer to achieve a 20-fold concentration.
SECTION: Medium fractionation
6. The sedimented fractions at each step were also collected and resuspended in sample buffer.
SECTION: Medium fractionation
7. All the fractions were analyzed by immunoblot.