We are new to exosome research as well. We are isolating them from patient serum. We are using a commercial reagent called Exoquick. https://www.systembio.com/microrna-research/exoquick-exosomes/overview

My understanding is that the reagent is probably some variation of older technique of PEG precipitation of viral particles. So it is likely one could make a PEG solution of the right concentration and accomplish exosome precipitation without having to pay huge amounts for the commercial version. 

If you are isolating exosomes from cells grown in tissue culture, I would think you will need to find some kind of serum free media as any serum in the growth media would contribute its own exosomes (i.e. cow exosomes from bovine serum).

All the best!

Hi Xiao-Gang Zhang

we are going to isolate exosomes from Mesenchymal Stem Cells.

Hello

Scientists have discussed the issues at this link :

https://www.researchgate.net/post/exosom_isolation_and_characterization?tpr_view=vaXfhf0q1g5EuoVjL8s4kGu31WuavOhGpana_2

And see these links 

Good Luck

http://www.lehmanns.de/shop/naturwissenschaften/33234115-9780128004975-mesenchymal-stem-cell-derived-exosomes

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4308409/

http://www.futuremedicine.com/doi/abs/10.2217/rme.12.61

http://cdn.intechopen.com/pdfs-wm/46601.pdf

http://www.ncbi.nlm.nih.gov/pubmed/27236691

Hello! We use ExoPrep reagent to isolate exosomes, for cell culture media we use ratio 1/1, of course, as was written above, it depends how rich of exosomes is cell line you use. Usually for one sample it is enough 1-3 ml of supernatant

thank you all for your kin help.

There are many ways you could go about the isolation, you can either stick with ultracentrifugation or using commercially available kits like exoquick, I heard it was also possible to isolate exosomes with ultrafiltration. this article here should be of use. It compares the different ways of exosome isolation.

http://www.journalofextracellularvesicles.net/index.php/jev/article/view/27031

You should also have some idea for exosome characterisation after this article, good luck.

The isolation method will depend on the amount of exosomes and also on the downstream analysis. We use nanosight (NTA system) to determine size distribution and concentration and it doesn’t work well when we use an isolation kit instead of serial ultracentrifugation.

It varies from cell to cell type. You have to do trial and error yourself and find out what will be the least number of cells secreting your desired exosome numbers and what will be the downstream experiment for your purpose. For instance, I seed 500,000 human trophoblast cells initially and after 48h, exosomal RNA is high enough to do RNA Next Generation Sequencing. If you are only interested in exosomes and not microvesicles (which I do not know the exact reason behind it), as the literature says, exosomes are 30-100 nm. You can first filter your culture media with 0.1 micron filter, then do exosome extraction with a commertial or traditional methods. If you would like to do it on a sucrose gradient, exosomes sediment at 1.13-1.19 g/ml. I hope this helped.

https://www.emdmillipore.com/US/en/product/Millex-VV-Syringe-Filter-Unit,-0.1%C2%A0%C2%B5m,-PVDF,-33%C2%A0mm,-gamma-sterilized,MM_NF-SLVV033RS