I have been attempting to extract exosomes from both frozen human brain tissue, and frozen or fresh mouse brain tissue. I've run into an issue where the final pellet produced by the exosome extraction kit, ExoQuick-TC, is not showing positive on a western blot for the exosome marker CD9, while my control exosomes from a former student are.

When I collected fractions from multiple stages in the extraction, I see a lot of them stuck in my first and second pellets. The first pellet was a 300xg spin to pellet cells and cell debris, and the second pellet was a 2000xg spin that should have pelleted apoptotic bodies.

As far as I understand, neither of those speeds is fast enough to pellet exosomes on their own, since they typically require ultracentrifugation if you aren't using a kit. I've troubleshooted the digestion step, increasing the concentration of the Collagenase type 3 that I was using to digest the tissue as it was purchased nearly 3 years ago and stored at -20C. Tripling the concentration from 75U/mL to 225U/mL helped the tissue digest much better than it had before, but I'm still seeing most of the signal in those first two pellets, and only the faintest signal in the enriched exosome pellet from the kit.

If anyone else has had luck using these kits to extract from tissue instead of culture media or plasma, I would greatly appreciate your advice!

Thanks for your time,

Ben Landon