Hi everybody,
I am going to optimize HUVECs cell culture condition in our lab to get high yield of exosomes but unfortunately we don't have NTA. Do you have any suggestion to calculate exosome concentration in conditioned medium?
Thank you.
Hi Fatemeh,
You can possibly try to measure just the protein concentration, for example by microBCA. But you have to take into account that this assay doesn’t discern between protein content of EVs and proteins in the medium or secreted by cells. That is why it is always good to process the unconditioned medium the same way as you do with the medium conditioned by your cells and use it as a control. Moreover, the results will also depend on the isolation technique. When you use density gradient ultracentrifugation, you will get much purer vesicles comparing to the differential ultracentrifugation but the protein concentration will be lower (because you will get rid of at least some proteins that are not of the same density as EVs) .
The same problem is with NTA or TRPS, these techniques are not able to tell the difference between protein aggregates and EVs, they just measure whatever particle that scatters the light. So, it is a kind of alchemy to tell how many EVs are actually in your sample and the best way is to combine several techniques like microBCA, Western blot and TEM,... You can read more about the recommendations for EV isolation and characterization in the new MISEV guidelines. Best, Jana
https://www.tandfonline.com/doi/full/10.1080/20013078.2018.1535750
no easy way; if you try to quantify EV protein etc- it might result in a huge error. We routinely use Nanosight, and there are 3 other instrument options on the market for quantification and sizing of nanoparticles
Yes, there is also TRPS (tunable resistive pulse sensing) that measures changes in the electrical resistance as the particle goes through the nanopore. But as the EV samples are usually not homogenous, the nanopores keep on clogging. The technology I like currently the most is MRPS (microfluidic resistive pulse sensing) from Spectradyne that uses microfluidic chips with filters so that the nanoconstrictions don’t clog that much. It is sensitive and you need just a few ul of sample. But anyway, none of these technologies are able to distinguish between vesicles and other particles/ protein aggregates.
Hi Fatemeh,
In order to quantify exosomes you can use NTA Analysis, including ZetaView instrument if you are interested. If you do not have the instrument, we offer it as a Service, at a competitive price. Let me know!
Julia
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