Dear Renjitha,

You can also use ultracentrifuge method to isolate exosomes.

And for characterization instead of TEM or Flow citometry you can use Nanosight analysis.

Best regards,

Marta Garcia

Thanks Marta

How do you go about Nanosight analysis? 

Also using the ultracentrifuge is not possible soon due to some facility restrictions. Hence looking for alternatives.

you are welcome! we can do a colaboration with your group, I could process your samples in the Nanosight Instrument.

About the kits also there is a new one from System biosciences and from Exiqon. I just tried Exoquick and 101BIO exosome isolation kits. Mostly I use Ultracentrifuge isolation protocol.

Let me get back to you next week Marta. By then my protocol and methods will be finalized. It would be great if we can collaborate.

once the exosomes are recovered with the Total reagent or ultracentrifugation or other method- we routinely use Nanosight LM10 to count and size them. This company is part of Malvern now. There are couple other instruments on the market, but i love this one

Dear Renjitha,

It is a pity you have no access to an ultracentrifuge since from our experience we can tell that using these commercial kits it will be very difficult, if not impossible, to get exosomes of satisfactory purity. We have recently compared both ExoQuick and Total Exosome Isolation reagent to (single-step) ultracentrifugation and a iodixanol density gradient, and all results indicated that only the latter provides pure exosomes. It's good to bear in mind that the choice of isolation method can severely bias your results.

http://www.journalofextracellularvesicles.net/index.php/jev/article/view/24858

Thank you Jan. I also understand that ultracentrifugation is the best procedure after talking to researchers in this field. Am trying out a collaboration. Thanks for your input. Will def read your paper!

Glad to hear that, I wish you all the best with the collaboration!

Hey guys, have a quick question. I am trying to isolate exosomes from serum for quantification and characterisation using nanosight. Do I need to ultracentrifuge to pellet the exosomes and then resuspend it again in filtered H2O to run through the nanosight? That seems like a redundant step based on my limited understanding.

In Jan Van Deun's paper:

- 300g: removes cells

- 10,000g: removes microvesicles

- 100,000g: pellet exosomes

Can I just dilute my serum 1:1 with PBS, centrifuge 300g to remove cells/debris, centrifuge again 10,000g to remove microvesicles, filter through 0.22um filter and then run the resultant supernatent (with appropriate dilution) through the nanosight.

It seems as though centrifuging at 100,000g just serves to pellet the exosomes based on my limited understanding. Has anyone looked at the supernatent after ultracentrifuging at 100,000g to see what's left behind?

Hi Matthew,

In my experience (which is predominantly with cell culture media), you will always have contaminants such as protein complexes when you only perform ultracentrifugation. A lot of them will remain in the supernatant and you will see these with NTA. Some will also end up in the pellet and are indistinguishable from vesicles because of a similar size and light scatter (as was the case in my paper). Because you work with serum, which is a more complex and viscous matrix than cell culture media, I would definitely perform UC with a washing step in a large volume to try and get rid of as much contaminants as you can. Maybe even consider diluting more than 1:1 before performing the centrifugations. Otherwise you won't know what you are looking at with the NTA. To be sure to have really pure EVs you would need to perform a density gradient in my opinion.

Hope this helps!

Jan