What have you used to EVs normalization from cell culture for functional assays (protein quantification, number of vesciles, number of producing cells, conditioned media start volume, etc)?
I would aprreciate any comments in that topic.
Hi there, although we haven't published a method yet, our lab is tossing around the idea of normalizing microvesicles by number. As long as you have a good flow cytometer in a biological safety cabinet, then you can keep them sterile. Careful though, not all flow cytometers/cell sorters can recognize small particles like EVs. You can tag your EVs with annexin V-FITC in high calcium buffer, then remove the calcium with a small amount of EDTA allowing the annexin to dissociate so you can use your EVs for cell culture.
Here are two papers that isolate EVs with annexin V:
http://www.journalofextracellularvesicles.net/index.php/jev/article/view/29159
http://onlinelibrary.wiley.com/doi/10.1111/imm.12493/full#references
Hope this helps!
Hi Maurício,
I would say It depends on your purpose. Protein content is not always proportional to the number of vesicles: cells under different stimuli increase the number of vesicles released in the medium but the protein content packaged in vesicles is reduced (it should be considered the quantity and quality of the EVs). Anyway, evaluating the number of vesicles is challenging (NanoSight helps). For functional assay I would suggest start using the number of producing cells for normalisation.
Hi Mauricio
As stated by Franco, the purpose is key because protein and active molecule MV contents vary with the cell origin and stimulus. Therefore protein content is a good approach only in one specific experimenal model making comparisons difficul among different cell types and stimuli.
So, MV number may be attractive (but you will have to consider the MV size distribution and will face the detection limits), phospatidçylserine dependent quantification is another way, since it s a a common feature exposed by MVs ( exluding exosomes) . For this you will need either annexin-5 (with calcium) and or enzymatic prothrombinase assay.
For normalization, number of producing cells are often used, with attention to the number of living vs. dead cells
Dear all, thank you for your time and answers. Flow cytometry would be excellent, but we don't have access to a powerful enough machine. In this case, I will use the number of producing cells for normalization.
All the best!!
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