Hello Nabil Aj

A) It is necessary to lyse exosomes before measuring their protein content.

I have a suggestion. Following exosome isolation, you may divide the exosome into two tubes.

1. One tube may contain exosome preparation in PBS for exosomal morphology, nanoparticle tracking analysis (NTA) and stability evaluation.

2. The second tube may be used for protein quantification (BCA assay) and Western Blot.

B) Sonication is a physical treatment based on bubble cavitation by ultrasound waves that promote a non-specific surface barrier disruption. When performed, sonication induces non-homogeneous disruption. Exosomes adjacent to collapsing cavitation bubbles get broken, whereas those located farther away experience a smaller local energy flux and are not lysed. So, sonication alone will not be sufficient for complete extraction of proteins. Therefore, you should use a chemical treatment accompanied by a secondary cell disruption method like sonication.

I suggest that you dissolve the exosome pellet in the protein lysis buffer of choice and pipette thoroughly, followed by vortex-mixing. To further lyse the exosomes, the exosome preparation may be sonicated 3x, 5 sec/cycle with vortex-mixing in between. Finally centrifuge the sample at 13000 x g for 5 minutes, and transfer the supernatant to a new tube for protein quantification and Western Blot analysis.

There is no standard procedure which you could refer to for protein extraction. In fact, many researchers try to create and adjust their procedure, and validate protocols.

The article attached below will be helpful.

https://chemrxiv.org/engage/api-gateway/chemrxiv/assets/orp/resource/item/64ff34a9b338ec988a4ad7d3/original/extracellular-vesicles-lysis-a-guide-for-the-release-of-biomarkers-to-be-used-in-cancer-diagnostics.pdf

Best.

@Malcolm Nobre thank you so much for the helpful information. For the lyse buffer, I only have Triton-X 100 and PBS. Is it enough to make the buffer and lyse the exosomes?

Also, should I use/buy exosome-depleted FBS to culture the cells before exosome isolation? If so, can I just centrifuge my current FBS without exosome-depleted?

Thank you.

You may add a physical disruption method and lysozyme to the Triton-X 100-based lysis buffer treatment to improve efficiency.

Generally speaking, the mentioned lysis buffer composition would be enough to lysis most of the EVs but it highly depends on the EV type you are working with. Therefore, a trial-and-error-based approach is used to find the best lysis buffer composition. RIPA is the far most considered recipe for EV lysis in the literature...

On the other hand, I do not have particular experience but I may strongly suggest using either exosome-depleted serum to ease the purification process. It would also be ok to ultracentrifuge the sample to remove EVs but some of the growth factors and necessary proteins/lipoproteins of the FBS may also be removed by this approach and could be assumed risky to implement.