I am using gradient ultracentrifugation procedure in order to do the same. Wherein I am using sorval A-641 rotor at 32900rpm (~120,000g ) for 2 hours for final steps. I am also finding some difficulties in dissolving the final pellet in PBS.
I have tried to check for the yield of exosomes in the literature, but I couldn't find solid evidences. As only few of them have stated the actual amount of starting material which they have used and the yield which they are getting.
Any help would be much appreciated.
Thank you !
Hey
I think this is something you should determine with your experiment since every cell type and stimulus will influence the the resulting amount of exosomes.
200 ug of exosome protein sounds a lot to me. I would give it a go growing your cells in 4 flask of 175 cm2 and then pooling it together.
Furthermore, what type of tubes are you using for the isolation? I am using a bell shaped ones in which the pipette doesn't fit...therefore I was having problems with my yield when I had to add small amounts to disolved it. I decided to resuspended in 1 mL, which increase my yield significantly, and then if a higher concentration was needed to concentrated with Amicon-100K filters.
Hope this help a bit, and good luck
Thank you David for your response,
I am also using bell shaped tubes for the final centrifugation steps which can hold upto 75 ml.
I read few roughly resuspend it very roughly or keep the pellet in PBS at RT for a while. I am just worried whether letting it stand in PBS at RT would affect the exosomes or not?
Can I know how much of conditioned medium do you use for isolation and how much yield of exosomes do you get?
Thanks,
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