I am trying to quantify the amount of exosomes in conditioned cell culture supernatant of monocytes with FACS analysis. Is it possible to perform a FACS analysis without an exosome purification step just by using the conditioned cell culture media?
I am not interested in any separation exosomes for further evaluation. I just want to know, whether exosomes are present in my conditioned media.
Thank you!
Hey lucian, do you mean exosomes between 30nm and 100nm or 100nm and 1000nm (also referred as microparticles?)
It's not so easy to separate noise from exosomes by flow (no quantification). But you can easily characterise them by coupling to antiCD63-coated beads (or other multi vesicular bodies markers such as tetraspanins) and then staining them with other fluorochrome conjugated Abs.
Yes, right. You need to couple them to some beads. But, its better to purify exosomes and then couple it.
Just for reference to this method:
http://onlinelibrary.wiley.com/doi/10.1002/ijc.28174/full
Thank you for your answers.
I will give you an short update. We have used Exosome Isolation Kit (Invitrogen) and thereafter antiCD63 coated-beads (invitrogen) for exosome detection. It worked quite good. We used anti-CD9 and CD63 as second ab. and got a dubble positive population in the cell culture supernatant in several indipendent experiments. Now we are trying to perform EM images - this is ongoing.
Dear Lucian,
maybe you can get in contact to Dr Gerhard Jungwirth, Kinderklinik, Medical University of Vienna. He´s got one of our NanoSight NTA instruments with fluorescence function and is characterising Exosomes as well. I am sure he can help.
Hi Lucian,
I agree with Dr. Bhat with regard to this question. We have tried it both ways and get better results from purification first.
its also possible now to use Dynabeads coated with anti-CD9, anti-CD81 or anti-EpCAM for evaluating exosomes in flow cytometry (Oksvold et al 2014. Clinical Therapeutics). let me know if I can help.
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