When I isolate MSC-derived exosomes using differential ultracentrifugation, I finally suspend the pellets in PBS. I pipetted the pellets up and down again and again until they almost dissolved in PBS. But every time I measured the concentration of exosomes by BCA, the products were always less than I expected. I did see the exosome pellets clearly, but I don't know why the yield of my final products was so poor. If anyone can offer advice, I'd appreciate that very much.
I don't know how much volume of supernatant of cell culture do you use to isolate exosome? What is your purpose when you measuring the concentration of exosome? BCA is used for measuring protein concentration, if you want to measure exosome, it's better to use Nanotracking analysis (NTA).
Nguyen Tien Cuong Well, I collected 50ml cell culture supernatant. I measure the concentration of exosomes in order to prepare different concentrations of exosomal solutions. I've learned the NTA in some papers, but here in my lab, there's no one using it.
Xiaoqin Li How much volume of PBS did you suspend exosomal pellet? Normally it would be 100-150ul. If you use too much you may not detect any exosome. I woud suggest you lysing your exosome by suspending your exosome with RIPA instead of PBS to see any difference since exosomes are particles and have sphere shape.
Nguyen Tien Cuong Thanks for your kind suggestions. Actually, I prepare exosomal solutions because I want to see the effect of exosomes on the cells I research. So it might not be a good choice for me to suspend them in RIPA. I usually use 200ul PBS for each 25ml-ultracentrifugation tube, and I'll try reducing the volume of PBS next time. Thank you again.
hi,
actually, the use of strong ion exchange principle for exosome isolation works very well. the zeta potential of exosomes are negative at the pH of 7 so they will bind quickly to a positively charged magnetic bead (Dynabeads Intact Virus Enrichment). The binding kinetics is very fast and capture protocol can be as short as 15-20 minutes for generic exosome isolation. The captured exosomes can be processed for e.g western blot or be eluted off with a counter anion such as KI.
kind regards
ketil
Xiaoqin Li If you want to do functional study with exosome I would suggest washing exosome with PBS right after ultracentrifugation.
Hi
From my long experience with exosome isolation and quantification, the best method to measure the exosome concentration is qNano, which is very precise method more than Nanosite technique. for the BSA I think it is method to determine the protein, SO if you would like to measure the protein inside the exosomes you have to rupture the exosomes, that let the protein to be free out side of the EXO. membrane.
Nguyen Tien Cuong Thanks for your kind advice. The yield of my final products has been much more steady.
If you measure the concentarion of exosomes using BCA, less concentartion of protein is normal because protein is encapsulated within the Exosomes. You try to lyse the exosomes by RIPA+ sonication then probably protein concentartion would increase. this is based on my experience.
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