I hope everyone is getting on with their research.
I am about to isolate extracellular vesicles (EVs) using frozen human plasma because I do not have access to fresh samples. Thus, I was wondering if anyone had experience with the isolation, characterization and biological properties of the EVs isolated from this source.
Any advice, thoughts or tips on the process will be very useful.
From the literature I have seen, most people do just work with fresh sample, however I few people have done it with frozen sample.
Thanks everyone,
David
Hi David,
You can work with frozen samples. Different studies showed that the EVs are stable in conditions such as freezing and thawing and can be recovered from frozen samples. However, multiple freezing and thawing can affect your reproducibility and recovery of EVs. Are your samples stored in -20 or -80? -80 leads to a better recovery and better preservation of cargos in the long term. You can check the quality of EV preparation by Nanosight, TEM, and Cryo-EM and compare your results with fresh samples. You should also perform a western blot for EVs markers in your samples.
I have done it. I started with fresh samples and then I repeated the isolation with frozen samples. Yes, you can work with frozen samples if stored properly. Although the reproducibility wasn't very satisfactory but again I faced some technical issues (in freezing the samples) so may be it was due to that.
It works perfectly well. Prior to ultracentrifugation, spin your thawed plasma sample at, for example, 6,000 x g for 20 min. to remove large aggregates of denatured proteins (if present) and large vesicle aggregates.
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