Hi,
I'm performing DLS on exosomes. For the most part, I'm getting very good, very tight reads consistent with the known size of exosomes! However, I do get a spike in the 1um range. I know there are no cells in my samples, so I believe this to be an aggregate of exosomes (or a very unlucky dust speck). How should I prevent this (the aggregation, the dust speck is kind of inevitable)? I've seen a variety of methods on the subject, but I'm wondering which is the most suitable.
Thanks.
Dear Colin,
Just a remark: I would not advise to use DLS on exosomal preparations because of their inherent polydispersity. You will get a nice peak, but it will not reflect the true size distribution of your sample. More reliable techniques are single particle tracking or resistive-pulse sensing. I've provided some further reading in case you're interested.
http://www.sciencedirect.com/science/article/pii/S0927776511002724
http://www.sciencedirect.com/science/article/pii/S0168365914008384
Hi Colin,
if you got the exosomes by differential centrifugations, the formation of aggregates are basically unavoidable unless producer cells are cultured in serum-free medium. For this reason, in many instances we prefer to purify exosomes by iodixanol gradients.
Best
Maurizio
Have you performed filtration (e.g. 0.2 um or 0.8 um)? Those 1um particles might be apoptotic bodies as well.
Dear Colin,
Just a remark: I would not advise to use DLS on exosomal preparations because of their inherent polydispersity. You will get a nice peak, but it will not reflect the true size distribution of your sample. More reliable techniques are single particle tracking or resistive-pulse sensing. I've provided some further reading in case you're interested.
http://www.sciencedirect.com/science/article/pii/S0927776511002724
http://www.sciencedirect.com/science/article/pii/S0168365914008384
We have used proteinase K and trypsin treatments to disperse exosomes when isolating by peptide affinity capture with varying degrees of success. This would however affect the extracellular protein components. Another method is washing with a high salt buffer. Hope this helps.
Gently sonicate exosomes prior to use. What are the physiological conditions that you are analyzing them in?
Alright, thank you for the tips. I'll try sonication.
And I don't ultracentrifuge anymore. Too high of a starting material cost for my cells. But thank you all!
How are you preparing your exosomes? Could the spike by microvesicles?
Trying the qEV columns by Izon. Under EM, they seem to be around the right size. But I have to test against exosome markers and Calnexin to make sure that these are exosome-like things.
Try 1% bovine serum albumin in the resuspension buffer after ultracentrifugation. Before doing this, consider how this may impact your analysis (bad for protein analysis, etc.).
PS: this also implies to use this solution as a background control for all downstream analysis. If your exosomes are from an epithelial source, you might also try a low concentration of EDTA to chelate Ca2+ and Mg2+ from cell surface homophillic adhesion molecules (e.g. Ecadherin, etc). Again, analyze the solution as a background control.
Are your exosomes fresh or frozen? Freezing often causes some aggregation. The occasional large apoptotic body is inevitable in any preparation and, as with most light scattering methods, DLS is known to over-estimate the number of larger objects. I would be careful if using sonication, as this can disrupt the membranes.
Normally for DLS centrifugation or filtration (even with large filter pore size) could help. However this may not be feasible for your sample. If adding BSA, filter (100nm or even 20nm) the BSA solution before adding as that can have aggregates as well. For low exosome concentration the BSA may show up as an additional peak (at about ~10nm diameter) in the intensity size distribution.
http://www.materials-talks.com/blog/2014/11/17/tips-tricks-for-nanoparticle-characterization/#exosomes
Colin, did you have some success with sonification? I had good results with filtration through a 200nm pore sized filter, but I'm afraid that I loose too many exosomes this way.
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