We performed the isolation of exosomes from the TMD8 cell line growing in exo-free FBS medium after it reached a confluence of 40 million cells/10ml. We used that mediium and tried isolating exosomes using the Total exosome isolation kit provided by Invitrogen, and further went on to perform Nanosight analysis. The data shows that we had exosomes, but the major portion of it was microvesicles. How do I get rid of microvesicles? Any inputs on this would be greatly helpful!
Hi Sneha!
How do you discriminate between exosomes and microvesicles using NanoSight? By the size only? What is the pre-analitical treatment of your samples?
You can try to centrifuge your samples (10 min at 14,000 x g should be sufficient) and afterwards ultrafiltrate the supernatants through 0.2 µm pore filters. This should eliminate most of bigger microvesicles (considering that they are 100-1000 nm in diameter). Just remember that this would not eliminate them in 100%, specially if you give too much pressure while filtrating.
However, I always use the pretreatment described above, and I get the mean peaks at NanoSight at 90-110 nm and no particles bigger than 300 nm.
There are also 0.1 µm filters available on the market, but be careful, because with the use of them you might eliminate also bigger exosomes.
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