I isolated exosomes from 0.5 ml of human plasma and serum with 2 kits from Invitrogen: Total Exosome Isolation Reagent (from plasma) and Total Exosome Isolation Reagent (from serum). I've tried to measure the number of partilces and their size by NanoSight, but I didn't succeed. What I could see was some "cloudy" picture and the screen was grey. From the other hand, I successfuly measured exosomes isolated by ultracentrifugation. I tried to dilute the Invitrogen-isolated samples 1:10, 1:100 and 1:1000, but without success. Does anyone know if there is some special protocol to do it? Or maybe it is not possible at all?
Hi magdalena,
Sometimes the remaining PEG present in the final pellet can make it difficult to visualize the exosomes on the nano sight. It might be a good idea to wash this final pellet using ultracentrifugation (100.000 x g for 70 min in PBS for example) and the try with this washed pellet.
Regards,
Eric
These protocols worked perfectly for us, we processed 100's of serum and plasma-derived exosomes on Nanosight LM10. I'd recommend processing lower volumes, eg 100 ul of plasma/serum, follow the Invitrogen protocol, then carefully remove all supernatant from the exosome pellet (can even re-spin to ensure you collect all droplets from the tube walls) and resuspend the vesicles in larger volume of PBS, eg 50-100 uL. Nanosight analysis requires a substantial dilution to get in the right range, as blood contains an extremely large number of vesicles
Hi Josina,
From my experience you can isolate plasma exosomes with both kits. However, they are not only exosomes that you isolate. You will find in the pellet a lots of proteins and all size of EVs. Measuring the amount of proteins by Bradford, I found that there was less proteins in TEI pellet than in ExoQuick pellet. The proteins and residuals of these kits chemicals can interfere with some downstream applications (me and my colleagues had a lots of problems with NanoSight). A part of larger EVs you can remove e.g. by filtration of your sample through 0.2 or better 0.1 filter, but this couse loss of exosomes as well. However, if it is important for you to isolate pure exosomes, maybe try some other methods e.g. size exclusion chromatography or density gradient ultracentrifugation.
Hope this helps somehow.
Hi Magdalena,
I have been using the Total Exosome Isolation (from cell culture) on my experiments and we have great results on NanoSight300 and TEM, but as you said it is not only exosomes that we isolate, sometimes microvesicles are present as well. I always follow the Invitrogen's protocol recommendations and then I dilute samples to 1:100 in PBS.
Regards,
João Pedro
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