May 16, 2025
Isolate Crude EVs by Ultracentrifugation
- Sierra Palumbos1,
- Erika Holzbaur1
- 1University of Pennsylvania
Protocol Citation: Sierra Palumbos, Erika Holzbaur 2025. Isolate Crude EVs by Ultracentrifugation. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnyommv5d/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: May 09, 2025
Last Modified: May 16, 2025
Protocol Integer ID: 218003
Keywords: EV, extracellular vesicle, ultracentrifugation, isolate crude evs by ultracentrifugation protocol, isolate crude ev, distinct compensatory secretory pathway, distinct compensatory secretory pathways in neuron, primary mouse neuron, autophagic stress, ultracentrifugation protocol, neuron
Funders Acknowledgements:
National Institute of Neurological Disorders and Stroke
Grant ID: R01-NS060698
National Institute of Neurological Disorders and Stroke
Grant ID: F32-NS129586
Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: MJFF-021130
Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: MJFF-15100
Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: MJFF-019411
Abstract
Protocol describing isolation of large (P20) and small (P100) extracellular vesicles isolated from primary mouse neurons. Protocol is used in "Autophagic stress activates distinct compensatory secretory pathways in neurons" by Palumbos et al., 2025
Troubleshooting
Grow Neurons- Until DIV 10-13
Isolate 5-20 million cortical neurons from e15.5 mice.
Plate neurons in 10 cm (3-5 million/plate).
At DIV7, ADD more media to existing. For primary neurons, do not remove any. For iNeurons, do regular media changes until 7 days prior to collection during which do not change it.
Grow neurons until DIV 10-13.
You can collect conditioned media earlier but you will have fewer EVs.
DO NOT CHANGE MEDIA.
Prepare for EV Isolation
Move rotors for ultracentrifugation to 4°C.
SW41 Ti to cold room and TLS 55 in fridge.
Filter 1X PBS.
Before collecting media, filter 1X PBS through a 0.22 micron filter.
Do this fresh every time!
Collect Conditioned Media
Using a Pasteur pipette, collect all conditioned media into a 50 mL conical tube and place on ice.
Wash and lyse cells.
This is important as you will need to normalize back to total protein, particularly if conditions were not consistent.
500 x g spin - Remove dead cells and debris
Take conditioned media and spin at 500 x g for 10 minutes.
Carefully remove supernatant and divide evenly within Eppendorf tubes.
If scaling up (e.g., 20 million for proteomics), do this in the ultracentrifuge.
Reserve pellet if comparing anything to dead cells/debris (e.g., histones).
20,000 x g spin - Isolation of Large EVs/ P20
Spin supernatant at 20,000 x g for 30 minutes at 4°C.
Collect supernatant using a 1 mL and 200 microliter pipette and place in 12 mL ultracentrifuge tubes.
Be careful not to disturb pellets - they should be visible.
Follow 100,000 x g spin below with collected supernatant.
Resuspend and combine all pellets (P20) in 1 mL filtered 1X PBS to wash pellets and remove remaining media.
Spin combined P20 samples at 20,000 x g for 30 minutes at 4°C.
Resuspend P20 pellet in 75-100 microliters of 1X PBS.
If using for Immunoblotting with no subsequent steps (e.g., Proteinase K treatment or iodixanol separation), immediately denature with 4X denaturing buffer and store at -20°C.
If using for NTA, calcein AM, etc., store at 4°C up to 2 days.
If doing further purification, store at 4°C up to 1 day before continuing, ideally do it on the same day.
100,000 x g spin - Isolation of Small EVs/ P100
Collect all supernatant from 20,000 x g spin and collect in 12 mL ultracentrifuge tube (Beckman 344059).
Fill tubes 80% with filtered 1X PBS.
Weigh correlating hanging buckets + tube + collected supernatant.
Opposite buckets need to be within 0.01 g! Ideally should show exactly the same readout on our balance.
Load buckets onto SW41 Ti rotor.
Make sure you check that BOTH hooks are in place. Buckets should freely swing. This is the most likely way a centrifuge will be imbalanced.
Put settings into XPN80 ultracentrifuge.
100,000 x g for 90 minutes at 4°C.
Start ultracentfigue when pressure is below 500 micron
Following spin, remove supernatant using a Pasteur pipette followed by a 200 microliter pipette and place in 15 mL tube for disposal.
The pellets are not visible, be very careful!
Resuspend pellet (P100) in 1 mL filtered 1X PBS to wash pellets and remove remaining media, place in microultracentrifuge tube (Beckman 347356).
Collect TLS 55 from fridge and place filled tubes in buckets.
Weigh tubes and buckets, again have weights equal.
Spin washed P100 samples at 100,000 x g for 90 minutes at 4°C using small ultracentrifuge.
Follow instructions above for first spin.
Resuspend P100 pellet in 75-100 microliters of 1X PBS.
If using for Immunoblotting with no subsequent steps (e.g., Proteinase K treatment or iodixanol separation), immediately denature with 4X denaturing buffer and store at -20°C.
If using for NTA, calcein AM, etc., store at 4°C up to 2 days.
If doing further purification, store at 4°C up to 1 day before continuing, ideally do it on the same day.