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My exosome pellet from ultra is invisible, am I aspirating it? Do they stick?

I isolate my exosomes from cell culture using ultracentrifugation 100,000 x g overnight. I spin 5mLs of serum free media which has been isolated from approximately 8 million cells over 18 hours.

I cannot see an adherent pellet and I am afraid I sometimes aspirate it as I get an "all or nothing" exosome signal.  I sometimes see a brownish "cloud" right at the bottom and it easily moves off the bottom. This is what I am afraid I am aspirating.

Our lab cannot afford the exosome isolation kits, so I would appreciate advice on ultracentrifugation.
asked Jan 28, 2014 in ExosomesTalk by kgoodmon

2 Answers

Ultracentrifugation typically recovers less exosomes vs the Total reagent. Taking into account that cell media has significantly lower exosome content versus body fluids sush as serum- the exosome pellet is invisible in many cases. Hopefully you are not losoing the precious exosomes but just isolate a very small amount that is hard to see. You can do Nanosight analysis or use some other easy readout- to confirm how much you got
answered Jan 30, 2014 by Vlassosv
I work with larger amounts of conditioned media (30ml at a time, 180ml total) and my pellet is very hard to see as well.

I find that I can see my pellet by holding the tube up in the light and looking for a raised bump on the surface. Often by pellet has a brown tinge, which disappears after a PBS wash.

I spin media at 100,000 x g but for 90 min, and my pellet does not easy wash off the side of the tube. I scraped it with a pipette tip when I resuspend it.
answered Mar 18, 2014 by navjay
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