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

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Renumbered during migration. The source had duplicate step numbers — two 7s, two 8s and two 10s — producing a 14-step protocol numbered as if it had 11 steps. The sequence below is continuous; the order of operations is unchanged.

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Materials

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All centrifugation steps should be carried out at 4°C. Keep tubes on ice.

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Protocol

  1. Transfer culture media to 30 mL screw cap centrifuge tubes and fill with 1X PBS until ~2 cm from the top — 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 × g for 45 minutes at 4°C to remove debris, using the Beckman Coulter Avanti J-E centrifuge with the JA-20 rotor (enter key number 20 into the machine). Press "door" to clamp the door down, then Enter + Start. Do not walk away until 12,000 × 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 tubes on ice — it helps to carve out a space for them.
  4. Carefully transfer the supernatant to a 50 mL 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 the liquid has been filtered, unscrew the syringe from the filter and take out the plunger. Repeat until all the supernatant has been filtered.
  6. When finished with the 30 mL screw cap tubes, wash once with ethanol and 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 × g (25,800 rpm for the SW-28 rotor) for 1.5 hours at 4°C. Make sure the red tubes are capped tightly so the vacuum works properly. Turn on the vacuum after loading samples into the rotor and wait until the vacuum level is below 20 microns before starting the run. Choose maximum acceleration and slowest deceleration. Press start and wait until the ultracentrifuge reaches top speed before leaving. Check back after ~10 minutes to ensure the run hasn't failed.
  9. Pipette off as much media as possible. We have found that pipetting gives higher numbers than decanting.
  10. Optional: add more media and repeat the process until you have gone through the media for each life stage.
  11. Do a final spin with 1X PBS.