I tried isolating exosomes from the TMD8 cell line- DLBCL cell line using the Total exosome isolation reagent kit by the invitrogen. We first grew our cells in exo-free FBS and allowed them to grow uintil they reached a confluence of ~40 million cells and used 10ml of our cells to isolate the exosomes. We performed a Nanosight analysis after the isolation, where we did observe exosomes but we also see a major portion of microvesicles. How do I get rid of microvesicles? Any inputs on this would be greatly appreciated!
Once you collect the cell conditioned medium, you remove floating cells (300 x g, 10 min), then apoptotic bodies (2,500 x g, 10 min). And if you centrifuge your sample then for 20-30 min at 20,000 x g you will get rid off the majority of microvesicles. After that you could apply Total Exosome Isolation kit to get exosomes (they will be contaminated with small microvesicles though).
Thank you very much for your response on this Igor. So, with this porotocol can I expect to see a good range of exosomes on Nanosight analysis? I am just expecting to see particles with an average size of the exosome range and the last time I got the mean size of the particle to be 209.7 nm and mode to be 129.2 nm.
Thanks,
the first step of the Total reagent workflow is pre-spin of the sample at low G-force, to deplete cell, debris and large vesicles. they you can add precipitation reagent and recover clean exosome fraction ~30-150 nm
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