I am isolating exosomes from 8 million epithelial cells in cell culture. I can very vaguely see a non-adherent "cloud" at the bottom of my tube after overnight centrifugation. Because it is so hard to see, I think I aspirate it from time to time. This "cloud" does have acetylcholine esterase activity. Should my pellet be sticking to the tube? Is there anything I can add or a certain tube I should use which would help it to stick to the bottom to prevent aspiration?
It's not necessary that you really see a pellet after exosomes isolation.It also can be that you don't see a pellet but that doesn't mean that there are no exosomes. Normally that pellet is quite good sticking at the bottom, its normally difficult to resuspend it because its so sticky.
A very cheep method of labeling is to treat your cells prior with any type of phenolic such as curcumin which will then stain the exosomes yellow which you can easily visualize by sight or by spec at 425nm. However, depending upon the type of rotor you use your pellet should be exactly where you would expect it to be. Good luck.
The pellet is not visible most of the times, but sometimes I can see a white small pellet. For fixed-angle rotors the pellet is on the side of the tube facing up. For swinging-bucket rotors the pellet is at the bottom of the tube.
Pellet is visible occasionally, as it is most glassy and very thin layer. Rest assured, if you are following the isolation protocol you WILL see the presence of exosomes in you desired fractions by either western blot or NTA.
Good Luck
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