After centrifugation-ultracentrifugation purification protocol (Thery et al protocol), I often noticed that my exosome preparations are composed of exosome aggregates. With the DLS analysis, the peak is between 150 and 1000nm and with electronic microscopy there is big aggregates of vesicles of the correct size for exosomes (30-150nm).
How could I help the exosomes to detach from each other without any damages for their structure? Do you think I could Vortex them?
Hi Laurent
I recently had a similar problem. What do you do with this pellet ?
You could try vortex, but Im not sure the extent of Exosome durability is well known.
I left mine at room temp for a couple of hours, attempting to resuspend periodically - the pellet was smaller after this, but still present.
What filter did you use and when did you filter ?
Regards
Jason
Hi Jason,
I solubilized the pellets in 100µl PBS by up and down pipetting during 2-3 minutes.
with the SAME samples I tried with or without 0,22µ filtering with a syringe before centrifugations.
The fact is that the DLS analyses showed that the preparation has bigger clumps with filtering than without filtering.
Another thing... When I left another 100µl PBS for a few hours in the tubes (after solubilizing the pellet) and I had a look with the DLS, there was nothing left in this volume with non-filtered samples and there was still big clumps (vesicles) with the filtered samples...
Any idea? Do you think filtering could facilitate/induce exosome clumping?
Regards
Laurent
I have experienced this problem and we have resolved this by incubating the exosome pellet in buffer for overnight at 4oC. In-fact, this is what we do to get the HIV particles back into solution following ultracentrifugation.
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