I have isolated exosomes from conditioned cell culture media . I have to go for its downstream applications and need to do the MTT assay. For that how to quantify the exosomes. Can we go with Nanodrop method ? We don't have the facility of Nanotracker.
Hi Bikash
HansaBioMed sells a kit called ExoTEST for exosome quantitation from human biofluids or cell culture media, based on an ELISA double-sandwich assay
Hi Bikash,
If you have isolated the exosomes and think they are concentrated enough for nanodrop you can also try quatifying total protein by BCA (nanodrop tends to give us variable results). We isolate exosomes routinely in the lab and referring to their concentration as total protein has given us reproducible results so far. If they are not concentrated enough you can try recovering them by ultracentrifugation (120,000 g X 2h) and resuspending them in a minimum volume of PBS. In our experience, exosomes from 100 ml of conditioned media concentrated and resuspended in 50-100 ul should give you a good reading by BCA. Best of luck on your experiments!
Thank You very much dear Salvador, It'll definitely help us to proceed further....
Another query, Should we add lysis buffer directly to the exosomes suspended in the PBS ?? We are not getting any visible pellet after ultracentrifugation according to the protocol by Thery et al . The UC tubes we have the capacity of 10 ml each and we are taking 8 tubes of concentrated media simultaneously.
Hello Bikash,
I agree fully with Salvador, BCA is best method to quantify exosomes as per my experience too as none of the methods work good (like BDFD and nanodrop) even the most sensitive instruments for protein determination gave us false positive results which involves resuspending your protein in some costly buffer. BCA works very fine. In my method I do protein isolation after concentration and then do quantification instead of measuring protein in concentrate directly as my work involves total protein n cell culture supernatant..
The isolated protein can be measured directly by BCA.
Hope it helps !
Hey Ajay
I have one question about the BCA protein estimation. How much of protein concentration do you expect to obtain if you use 40ml of the conditioned medium?
I am getting 6ug/ml from BCA kit after using 40ml of starting medium and I am suspecting it to be very low for any further assay.
Any help would be of great use.
Thanks
Hello Vasu,
I would suggest you to concentrate your CM further as per the method described above and do protein extraction. 40ml starting volume is good to give decent concentration of protein. After protein extraction, pls don't dissolve extracted protein in 1ml PBS (I doubt you haven't done that). According to pellet size, try to dissolve in 100-200ul of PBS or suitable buffer. You will get a good protein concentration there. Here you will have freedom to dilute your sample if you obtain a high protein concentration.
Hope it helps!
Hey Ajay,
Thank you for your quick reply. I resuspended the pellet in 500ul of PBS at the final step, seems too much as compared to what you have suggested(100-200ul), I will surely try to use less volume next time
I have one more small doubt. You have mentioned about protein extraction above.
I did the protein estimation directly after resuspending the pellet in 500ul of PBS at the final step. How much fold difference in the protein reading do you expect to see while using exosome suspension directly without lysis or protein extraction and one after protein extraction?
Thanks in advance!
Hay Vasu,
Since exosomes are membrane bound vesicles harboring protein, RNA, DNA, miRNA including ncRNA etc. so I think getting proteins out of the exosomes by lysis or protein extraction is really important. That might be another reason for your low protein concentration. Please extract protein, that's hardly a 30min protocol. You will find that easily on internet. Since exosomes contain many things as explained above and if you intend to treat something with exosome proteins, then treatment of exosomes may not be sole protein treatment. For protein treatment, you need to extract protein and then use it. In our case, we did animal treatment with isolated protein. You need to take care of that.
Hope it helps!
Thank you so much Ajay !
I will try your suggestions.
hope they work out well.
Thanks..Please update about your results after implementing these suggestion.
Cheers!
You can quantify using DLS for the size of exosoma and Nanodrop for the absobace at 280 nm to define the number of particles . I use a scale made with protein BSA nanoparticles.
Hi Ajay Kumar ,
Do you have a protocol for lysing exosomes with RIPA buffer? I have resuspended my exosome pellet in 50 ul of PBS, and I want to measure the concentration with BCA assay. How long do you incubate your exosome with RIPA? Is it at room temp or in ice? Do you sonicate your sample in RIPA?
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