Hello,
I am trying to isolate exosomes from cell lines of DLBCL. I cultured my cells in medium with regular FBS and once they had reached confluency, I transferred the cells into exo-free FBS medium and allowed it to grow for 72 hours after which I performed a series of ultracentrifugation spins.
a) 300*g for 10 minutes at 4 degrees.
b) 2000*g for 10 minutes.
c) Ultrafiltration using a 0.2 micron filter.
d) Supernatant was transferred into a new tube and 100,000*g spin for 90 minutes was performed.
e) Supernatant was decanted, leaving behind 1ml to which 9 ml of fresh PBS was added and another 100,000*g spin for 90 minutes was performed.
f) Supernatant discarded, and to the pellet, 200 ul of ice-cold fresh PBS was added.
This mix was prepared for Nanosight analysis, but the size range obtained is always >150 nm (expected size) [around 160-175 nm]. Is there any other step that I should include /perform to eliminate the bigger microvesicles?
Any suggestions would be appreciated!
Thanks,
Well, I would start by asking WHY you want/need to select for the smaller populations of vesicles? Are your molecules/signaling functions of interest necessarily associated with small EVs?
The protocol looks solid for isolating EVs in general. If you want to focus on "exosomes", you could resuspend the pellet in PBS and load it onto a density gradient. This additional purification step allows you to extract EVs with a specific floatation density, which is one of the characteristics used in the attempt to define what "exosomes" actually are.
A less stringent approach would be to isolate EVs from (pre-cleared) supernatant by Size-Exclusion Chromatography (SEC). This method separates EVs by size and, as denstiy gradient centrifugation, has the added benefit of removing soluble proteins as well.
Hello, the method that would give you the purest exosomes is the density gradient, but if you can't/don´t want to do it, you could try and include a 20.000g centrifugation step before your step d), as the 0,2um filtration usually allow some bigger vesicles to pass through it.
Try it and see if you can obtain a smaller size range.
Thank you very much for providing suggestions on improving the process. Could anyone post an optmized process to perform a sucrose gradient?
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