I am trying to get exosomes from MDCK and Balbc 3T3 lines using the Exoquick-TC kit from SBI. At first I tried with T 75 flasks (that means I collect 6 ml of culture media -serum free-/flask), but now I am working with T150 flasks in order to can seed more cells and get more exosomes (I collect 12 ml of culture media -serum free-/flask).
The thing is that the pellet I can see is really small in comparision with the ones they show in the user manual of the product (EXOTC10A-1). The picture (in the user manual of the product) are exosomes from cerebral spinal fluid and in my case I am trying to get it from cells, so it is a very important thing. I think the problem is the amount of starting material. I know that with this cells lines people works with a really big amount of flasks (eg x10 T150 flasks) when they want to purify them by ultracentrifugation, but I thought that with the kit I were going to be able to get a pellet with less amount (and this is the reason I have started with less flasks).
I am going to do a WB in order to see if these little pellet really contains exosomes or not but I would like to know if some one has the same problem as me. I see the little pellet using x1 T150 and now I have x3
T150 wich culture media I am going to purify tomorrow).
Thanks so much
cells secrete rather small amounts of exosomes- compared to body fluids such as serum. sometimes you dont see the pellet, unless you process a very large volume of cell culture media
Hi Beatriz,
in my hands it is very difficult to see any pellet (I am working with ECFCs and using the ultracentrifugation methods) even using 200 ml of conditined medium. Just for your information, I used the kit in the past trying to get exosomes using less quantity of media and I saw some pellet but when I checked the exosomal markers those were not present...so I keep the classical ultracentrifugation method in my protocol. Good luck!
Thanks for your answer Alexander Vlassov. At the end I were able to see pellet from the x3 T150 flasks using the kit. Soon I am going to try by ultracentrifugation too, but it seems more difficult to see the pellet by this method (as far as I have read in bibliography).
Thanks for your answer Jose Luis Viñas. I finally got a little but clearly visible pellet from x3 T 150 flasks using the kit (that means 36 ml of culture media). This week I am going to do a Western Blot in order to check if there are present some exosomal biomarkers. The next step is to compare the purity of these exosomes with the ones obtained by ultracentrifugation in order to choose the best one. Thanks so much
Pues mucha suerte Beatriz!, Just let me know if you need further help or information.
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