Exosome Isolation from Parasite Culture Media

This protocol is used to purify exosome-like vesicles (ELVs) from nematode culture media. Filarial nematode culture media is typically collected every 24 hour for eventual ELV isolation.

Materials

  • 1X PBS
  • 0.22 µm syringe filters
  • Ultracentrifuge metal tubes
  • Beckman centrifuge tubes

Protocol

Note: All centrifugation setps should be carried out at 4°C. Keep tubes on ice.

  1. Transfer culture media to 30 mL screw cap centrifuge tubes and fill with 1x PBS until ~ 2cm from the top of the tube (do not fill to the brim). If you do not have an even number of tubes, fill a screw cap tube with water. Balance your tubes using PBS on the scale.

  2. Centrifuge media at 12,000 x g for 45 mins at 4°C to remove debris (Beckman Coulter Avanti J-E Centrifuge). Use the JA-20 rotor (enter key number: 20 into the machine). Press “door” to clamp the door down. Press Enter + Start to start the centrifuge. Do not walk away until 12,000 x g is reached and the machine is not making any strange noises.

  3. Be very careful when removing the screw cap tubes from the centrifuge to avoid dislodging the pellet. Put the screw cap tubes on ice (tip: carve out a space for the tubes in the ice).

  4. Carefully transfer the supernatant to a 50mL conical tube without disturbing the debris pellet using an electronic pipette.

  5. Filter the supernatant through 0.22 μm filters using a 10 mL syringe into a Beckman centrifuge tube. Take out the syringe plunger, screw the syringe onto the filter, use an electronic pipette to fill the syringe with supernatant, and gently push down to filter. When all of the liquid in the syringe has been filtered, unscrew the syringe form the filter, and then take out the plunger. Repeat until all of the supernatant has been filtered.

  6. When finished with the 30mL screw cap tubes, wash with ethanol once and then once with PBS.

  7. Place the Beckman ultracentrifuge tubes into the red ultracentrifuge tube holders. Fill these tubes to the brim and balance with PBS.

  8. Centrifuge at 120,000 x g (25,800 rpm for the SW-28 rotor) for 1.50 hours at 4°C. Make sure the red tubes are completely capped tightly in order for the vacuum to work properly. Turn on the vacuum after loading samples into the rotor. Wait until the vacuum level shows it is <20 microns before starting the run. Choose the max acceleration and slowest deceleration before starting spin. Press start and wait until the ultracentrifuge has achieved top spin speed before leaving. Check back on the machine after ~10 minutes to ensure the run hasn’t failed and stopped spinning.

  9. Pipette off as much media as possible (we’ve found that pipetting gives us higher numbers than decanting the media).

  10. Optional: add more media and repeat the process until you’ve gone through the media for each life stage.

  11. Do a final spin with 1X PBS.

  12. Remove all but about 4 mL of media with the electronic pipette. Use a p1000 to remove most of what remains, leaving only ~ 1.5 mL.

  13. Wash the walls by pipetting. Transfer into a 1.5 ml ultracentrifuge tube.

  14. Optional (to concentrate ELVs): Centrifuge at 55,000 rpm for 2 hours at 4°C. Remove all but ~25 uL.

ELVs can be quantified using Nanoparticle Tracking Analysis (NTA)