Hi Milad,

I would try reduce incubation time with agent

and/or

reduce the concentration of agent and extend incubation.

If you really wanna stick to that concentration and incubation time, which is toxic to cells, options are narrow. (in this case, major problem still persists, if your cells are sick by the treatment, why you wanna compare, also absolutely depends on your study concept)

what you may can try to (at different time points or dose dependently):

1. isolate exosomes from both group of cells (assume from culturing medium) and get exosome number use nanosight (or FACS using beads you can visualize your exosomes)

2. at same time determine the viable cells number in both group

3. now you can normalize exosome numbers/alive cells (however, keep in mind you have some exosomes released from dying cells)

4. finally see if, alive cell number correlates with an exosome number (if your agent enhances exosome secretion your result might be reasonable)

5. then compare treatment and control

If you have no FACS or nanosight available (or even better in addition to that), you can exogenously express exosomal marker protein fused to fluorescent protein, in your cells. After collection of exosome do immunoblot using antibody against your marker protein like GFP, then normalize to alive cells. (If you go pubmed there are plenty of papers regarding fusion protein expressing vectors)

Goodluck,

Exosomes are 40-200 nm membrane vesicles, present in a wide range of body fluids, including blood, urine and saliva. Exosomes reflect the physiological state of their cells of origin and consequently provide a rich source of potential biomarkers. However, the identification and quantification of exosome in clinical samples remain challenging. In this study, we established a highly sensitive and rapid analytical technique for profiling circulating exosomes. We call this assay “ExoScreen” and used ExoScreen to detect cancer-derived exosomes in human clinical samples. We found that CD147 and CD9 double-positive exosomes were significantly higher in serum from colorectal cancer patients (n= 194) than in serum from healthy donors (n = 191).

Isolate, then do NTA for exosome concentration changes, and additionally can try protein assay (RC/DC for example) to see if exo-protein is effected by treatment. Check viability with Trypan or do a full MTS assay to have better judgment on proliferative effects of changing exosome release. If there is an effect on viability this could be from multiple things, including but not limited to, toxicity of compound at certain concentration to cells or the outcome of preventing exosomal release of compounds that may have an anti-proliferative or cell killing effect. Hope that is helpful

Thank you all for your detailed answers.

Dear Phillip,

I have tried protein assay using the BCA kit; however, I think this test would not be so accurate due to the following reason:

At the final stage of exosome isolation when the exosome pellet appears, I have to discard the supernatant and then resuspend the pellet in certain amounts of solvent (i.e. PBS in my case) which is a really delicate work since if any small amounts of supernatant remains on the pellet, it would change the final volume and consequently the concentration of my exosome solution which will differ among groups making it kind of impossible to compare concentrations.

Do you have any recommendations to solve this?

Hi Milad,

You bring up an important and valid point. I have encountered this similarly where I acquire data that suggests no difference in protein concentration (perhaps due to your stated problem with variable supernatant volumes) but do see significant differences in particle concentration and miRNA amounts. I think that NTA is the most robust way to accomplish this, but if protein is altered significantly enough you might still be able to see that change with BCA. Best of luck with your work.

Dear Phillip

Thabk you so much for spending your time posting on this question of mine. So helpful

All the best

Hi Milad,

To address the variable pellet resuspension volume, this is what I would suggest to get around that problem. Let's assume that you have to resuspend your pellets into 100 uL of PBS, and that the largest error you could expect from the issue you describe is, say, 20 uL extra (something you would determine from experience).

So, in this case, I would instead resuspend everything in 80 uL, and then go in with a micropipette and measure the actual volume contained in the tube. Use a 200 uL pipette, set it to 80 uL, and aspirate the whole sample. Then, you should see the extra volume still in the tube, so while still holding the pipette tip immersed into the leftover sample, increase the set dial on the pipette until there is no more sample to aspirate. The reading on your pipette is now the actual volume including the extra volume! Expel back into the tube and move on to the remaining samples. Calculate how much additional volume you need to add wherever needed so that all the samples are 100 uLexactly. This works whether you are resuspending cells, exosomes, protein, whatever, and I always do it when I have to resuspend in a specific volume that is much much smaller than the original centrifugation volume.

Just be aware that, due to pipetting error and pipette tip liquid retention, there will still be some error between your samples. However, this method will help you eliminate large errors that are inevitable when trying to resuspend pellets in very small volumes. Beyond that, simply repeating the experiment multiple times will help average out the error from the rest of the procedure, which should be much smaller. Good luck!

Dear Menelaos,

Thank you so much.. your answer kind of included all I need.. pretty useful