Did you confirm that you have the exosomes isolated and in enough amount? You can do this using Nanoparticle Tracking Analysis (NTA).

I isolated exosomes from MDA-231 cells using the same method as you did. Also, I tried a more reliable method using combined ultracentrifugation followed by ultrafiltration as follow:

Briefly, the conditioned medium was centrifuged at 600g for 30 min to remove cells and debris. The supernatant was centrifuged again at 2,000g for 30 min to remove apoptotic bodies. Microvesicles were collected by ultracentrifugation at 20,000g for 60 min.

Supernatants were filtered through 0.22- µm membrane filters (Thermo Scientific) with pressure to remove large vesicles.

Exosomes were collected by size-based EV isolation method with modifications [34] using 50 nm membrane filters (EMD Millipore, VMWP02500) with holders (EMD Millipore, SX0002500).

You can find the full protocol in our paper here

https://mct.aacrjournals.org/content/early/2019/08/28/1535-7163.MCT-19-0299

I will be happy to answer any more questions you have.

Thanks @SherifIbrahim. Actually I confirmed that I have vesicles both by NTA and by electron microscopy. Thats why I think its a problem with the quantity of vesicles secrted by MDA231 Cells. Maybe they have les RNA than other EVs

But thanks, I will check on that paper.

180 ml of media is plenty, and Qiagen kit should work for this input. Not sure what went wrong. we routinely use Total exosome RNA and protein purification kit (Invitrogen), for exosome preps from cell culture and misc body fluids

180ml of medium es very little. MDA-MB-231 is a poor cell line of exosome delivery. In my lab we using since of 500ml of medium FBS-free for protein or RNA extraction. Between the methodos used for exosome isolated, exo-spin or ultracentrifugation are very good methodos, but in both case is necesary at least 500ml of medium.

Between the methodes used for RNA isolated, I use at least 20ug of exosome and (1) Trizol variant to enriched in miRNA or (2)

Total Exosome RNA & Protein Isolation Kit (Invitrogen). the latter allows librery generation of miRNA or extraction protein/RNA total.

Thanks Emanuel,

But I hace questions. I'm definitely able to obtain 20 ug of exosome proteins from 180 ml conditioned medium, and I have used more than 20 ug of exosomes to obtain RNA (or microRNA) but I just can't

I have not try with trozo yet, but I can try after quarentine. Maybe I need to use all 500 ml of medium to collect exosomes and use all of the to obtain microRNA.

Thank you

Hi, i didn't try it yet, but i was told trizol extraction works better.

i will start working with the same cell line. if I may, i have a question about the medium you using, is it a free media or advanced one? and how many time incubation for the conditioned medium?

thank you

Hi Nait Eldjoudi Amina

I have not used trizol, but I read that Trizol LS works well.

Regarding to the media, we are using DMEM/F12 growth medium + 10% EV-depleted FBS. We depleted EVs by ultracentrifugation.

And we are collecting conditioned medium 48-72 hours after the seeding

Tell me how are you doing

Eduardo

Eduardo Duran Thank you for replying, I just started my thesis project, so when i checked MISEV 2018, they recommand serum free media or if not possible UC of FBS for like 18h, so i went with serum free media option in my first try with 10 dishes 100mm for 24 hours, but i got nothing. some recommand to me to use advanced medium with 10 flasques T150, and collecting the conditioned medium each 24 hours starting at 50% confluency till 80% changing the media evryday. am gonna try this and see ...

tell me what you think about it ^^

Amina

Hi Amina Nait Eldjoudi Amina

Yes, you are right, they recomend optimem I think, but we don't have that right now. So we are UC the FBS at 100.000 g for 16-18 hours and then filter it through 0.22 um filters to deplete it from EVs.

In the case of MDA231 cells I normally use UC or the exo-spin kit to obtain EVs. I use 1-1.5 million cells in 100 mm plates (or T75 flasks) to collect the conditioned medium after 48 hours. So, normally I collect 80-90 mL and the cells are in a ~70-80% confluency. Then you can use UC or the kits (if you use kits you can concentrate the CM with 100K Amicon filters first).

The yield is better using this kit compared with UC in terms of particles (but maybe you will obtain more contamination with proteins).

How do you know that you got nothing? Are you checking by western blot? NTA?

I think with those conditions you should get particles, maybe not so much because (at least in our hands) this cell line does not produce so many exosomes compared with other tumor cells, but at least you should have seen them you in the NTA.

Cheer up I hope I helped you

Thank you again Eduardo Duran your post help me a lot. I actualy didn't check with NTA, since i wanted to do a western blot i need much more particles, I tried again with 200 ml CM but i had a very low protein concentration that can't do for a WB; maybe i shoud go with 500ml CM as Emanuel Jeldes did; or do multiple isolations and pull them together, but in this case i need to find a way to concentrate my proteins.

Thank you

Hello Nait Eldjoudi Amina

Well, you could do that, I mean trying with multiple isolation, but I still think its wierd that you are obtaining low protein amounts.

In my case, using 100 ml of CM and using ultracentrifugation to purify EVs I obtain between 2-4 ug/ul protein concentration (resuspended in 200 ul filtered and sterile PBS).

How many cells are you plating? What is the confluency when you collect CM? Are you using optimem or medium suplemented with EV-depleted serum?

Hi Eduardo Duran ,

I don't understand what's wrong either. I had 10 flasques T175 per condition, and i used serum free medium, collected 100ml after 24h at 60% confluency, then changed medium and after another 24 hours i recollected another 100ml. but then (48 h) i was at 90% viability so i stoped collecting (i counted at final 40millions Cells).

Do you think you can send me the protocol you working with so i can check what's wrong?

thank you very much

Yes, no problem Nait Eldjoudi Amina

Give me an e-mail address and I will send the protocol.