Mar 03, 2025
Extracellular Vesicle Isolation
- Emily Tabaie1
- 1University of California, Riverside
Protocol Citation: Emily Tabaie 2025. Extracellular Vesicle Isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldr4eng5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 03, 2025
Last Modified: March 03, 2025
Protocol Integer ID: 123711
Abstract
These steps are used to isolate extracellular vesicles from primary neuronal cell cultures. It uses a combination of centrifugation and a ExoEasy Kit from Qiagen for isolation.
Extracellular Vesicle Isolation
Extracellular Vesicle Isolation
Do all steps in the hood and be sterile
Grow Neurons and ME49 B7 parasites separately
Change Neuron media every 3 days and save at -80°C
At Day 9 of the neurons purify the parasites and infect the neurons with an MOI of 0.5
After infection wait 3 hours and then change half of the neuronal media
Continue change and saving the neuron media until the neurons die
Once all the supernatant is collected and filtered centrifuge at 500 x g for 15 minutes at 4°C
Take out the supernatant and ultracentrifuge it at 15,000 x g for 20 minutes at 4°C
Use the exoEasy Maxi Kit (Qiagen 76064)
Add 1 volume buffer XBP to 1 volume of sample and mix well by gently inverting the tube 5 time
Let the mixture warm up to room temperature
Add the sample/XBP mix onto the exoEasy spin column and centrifuge at 500 x g for 1 min at room temperature
Discard the flow-through and place the column back into the same collection tube
Add 10 mL buffer XWP and centrifuge at 4,000 rpm for 5 min at room temperature to remove residual buffer from the column
Discard the flow-through together with the collection tube
Transfer the spin column to a fresh collection tube
Add 400 μl of Buffer XE to the membrane and incubate for 1 min
Centrifuge at 500 x g for 5 min at room temperature to collect the eluate
Re-apply the eluate to the exoEasy spin column membrane and incubate for 1 min
Centrifuge at 4,000 rpm for 5 min at room temperature to collect the eluate and transfer to an appropriate tube (not supplied)
Aliquot out the EVs and store at -80°C
Run NTA on the EVs the same day before freezing EVs