I am trying to purificate Extracellular vesicles (EV) from BOEC conditioned media by coloumn of chromatography, then i want to charachertize the purified EV by flow cytometry. However i have seen in some publications, that it is recomended to treat the sample of EV with Aldehyde/Sulfate Latex Beads, (Invitrogen), before to analyze them with cytometry, so i would like to know:
What is the exact role of these beads in the cytometry assay
and if somone had sucessfully charactherised the EV by cytometry without previous incubation with those beads ?
thank you so much
Hallo Meriem.
EVs (50-100 nm) are usually too small to see them in a conventional cytometer or they are hidden in the background. However, their membrane contains proteins, that can be conjugated to Aldehydes: The Amino moieties of the lysins react with the the Aldehyds to form a stable Imine (not too stable in acidic conditions!).
The sulfate is there for having a negative charge, keeping particles in suspension and suppressing non-specific binding. If you conjugate smaller particles to bigger ones (The Latex = Polystyrene beads are around 0.02 - 16 µm depending what you need) you can actually see them in the scatter-plots. Also they have a high refractive index, your EVs might not have- so the scatterings is also higher. So basically, they are there to make the EVs visible and stainable:
If they are attached to a bigger particle, you can fluorescently lable surface proteins of the EV using an labeled antibody (e. g. FITC- labeled). Then you can Quantify (or sem-quantify) how much protein is on the surface.
If you can separate them (eg. with SEC from other particles in the mixture ( cell fragments and so on) you could also measure size distributions via light scattering (DLS or SAXS)- but you might have a high "background"
Here is an article, that describes some procedures:
Article Improved Characterization of EV Preparations Based on Protei...
I hope that helped you.
Best regards
Peter
Hallo Meriem.
EVs (50-100 nm) are usually too small to see them in a conventional cytometer or they are hidden in the background. However, their membrane contains proteins, that can be conjugated to Aldehydes: The Amino moieties of the lysins react with the the Aldehyds to form a stable Imine (not too stable in acidic conditions!).
The sulfate is there for having a negative charge, keeping particles in suspension and suppressing non-specific binding. If you conjugate smaller particles to bigger ones (The Latex = Polystyrene beads are around 0.02 - 16 µm depending what you need) you can actually see them in the scatter-plots. Also they have a high refractive index, your EVs might not have- so the scatterings is also higher. So basically, they are there to make the EVs visible and stainable:
If they are attached to a bigger particle, you can fluorescently lable surface proteins of the EV using an labeled antibody (e. g. FITC- labeled). Then you can Quantify (or sem-quantify) how much protein is on the surface.
If you can separate them (eg. with SEC from other particles in the mixture ( cell fragments and so on) you could also measure size distributions via light scattering (DLS or SAXS)- but you might have a high "background"
Here is an article, that describes some procedures:
Article Improved Characterization of EV Preparations Based on Protei...
I hope that helped you.
Best regards
Peter
Hi Meriem, though you had your answer, but following links have some appealing information respective to your query and might be some more helpful if you look at the scientific explanation about these beads.
https://www.thermofisher.com/order/catalog/product/A37304
https://www.thermofisher.com/order/catalog/product/S37227
Sincerely....
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