I want to isolate MVs from colorectal cancer patient plasma. Can you advise which isolation method is the best for proteomic analysis? Ultracentrifugation at 100k g or filtration using 0.8um cellulose acetate membrane filter? I am interested in isolating larger MVs (100-1000nm) rather than exosomes.
Our lab uses a series of centrifugation steps to isolate different vesicle fractions from conditioned medium. An initial spin at 2,500 g to remove apoptotic bodies, followed by centrifugation at 12,000g to isolate micro vesicles (pellet). We then use the supernatant from that fraction to isolate exosomes and larger protein aggregates. I do not know if this would be useful for extraction from plasma.
Thank you, Avraham!
Yes, according to some articles, they do 2-step centrifugation at 1500-2500 g and at higher speeds up to 18 000g for plasma as well. However, you need to avoid all the junk proteins in plasma which could interfere MVs proteins. So, I thought maybe filtration method is better?
Immuno-magnetic separation may also be envisaged especially if you know good markers that came from the cells of origin (colorectal cancer cells) and went on the MVs serface (not all Ags from the cells of origin go onto MVs !). Suggestions may include EpCAM (for epidermoid cancer), EGFR-2, some cell-surface expressed cytokeratins, CEA, ... and many more. But you have to check with some models (e.g. colorectal cell lines in culture) or a good survey of the litterature (look for J of Extracellular Vesicles - JEV and ISEV congress abstracts) that the derived MVs do indeed express such cell-surface Ag. Best if you can get proofs using flow cytometry of MVs that such or such Ag is indeed present (if not familiar with that approach, try first CD41/ AnnV on Platelet MV which are far the most represented MVs inplasma). Open for collaboration in this field? We have some experience with both standardized FCM analysis of MP/MVs (not exosomes) and IMS of MP/MV. Hope that helps. (note: will be absent for a few weeks). Philippe
In my view size base discrimination will not be valid. Using force filtration might create artificial vesicles (or rupture them ) and in gravity base filtration one may saturate the pores and you will not recover all MVs. I think UC would be good way to getting most of MVs. Chk the following link
http://www.journalofextracellularvesicles.net/index.php/jev/article/view/20677
Thank you, Ganesh! I will consider your suggestion during experiments.
I agree with Ganesh. I would definitely stay away from filtration. I know many people use it. But there are no detailed studies of what squeezing vesicles through a certain size pore does to the vesicles themselves. It lipid matter. I am quite skeptical that a bigger particle would be reshaped into a smaller one, rather than stopped from passing through, just like when extruding lipid vesicles made artificially.
Ultrafiltration is quick and simple, but results in rather dirty prep. Ultracentrifugation can do a much better job, but it requires training and takes up to 30h - with sucrose gradient.
I agree with Alexander. However, I would also add that in my experience MV recovery (as approximately estimated by FACS analysis) seems to be higher with ultrafiltration than with ultracentrifugation. This is the reason why we use a combination of both. Can anybody confirm? Of course, the choice of your method also depends on your aim, i.e. which is the degree of purity you need for each specific application?
I'm working on a combined ultracentrifugation combine with a filtration protocol right now. One of the things I have read in several studies about filtration, and sort of the point Virginia was making, is that exomsomes are lipid molecules and can clump together causing difficulty going through poor openings. Further, exosomes can clump together with microvesicles making difficult for both to get through pores. However, recent evidence indicates that if you sonicate your samples (after removing cell debris), you can break apart the clumps and improve the efficiency of filtration. I'm going to start trying to incorporate a filtration step just to get "cleaner" samples in the future. That being said, in the past we have used just centrifugation steps for exosome isolation and had no problems.
http://www.ncbi.nlm.nih.gov/pubmed/24336651
Ultracentrifugation is the Best solution to obtain EVs large and small. Other methods are possible but there are mistake or detrimental effects, for example sonication give a new formation of vesicles from membrane.
You can centrifughe at 2000xg for 10 min to eliminate cells and cellular debris, then centrifuge supernatant at 10000xg for 30min to obtain a pellet of microvesicles, after centrifuge super. at 100000xg for 30min to obtain a exosome pellet.
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