I am looking to sort exosomes via tissue-specific surface markers, using antibodies. Fluorescence-assisted vesicle sorting be carried on the same equipment used for FACS?
Hello Mohamed Yousef,
it depends a bit on how big your Exos are. If they are in the usual 50-100 nm range, it will be very hard. You have a large background in the scatter detector in this size range (usually a threshold is set to exclude particles in this range from analysis/FACS). In the description of your cytometer it is stated what the minimal size detection is. Flow-rates need to be optimized, so that you have a very think core-stream. Also the sorting is not trivial. I cannot imagine, you have only 1 Exo/drop in the sorting, they are too small, but you can enrich them. As I told you in the manual of your cytometer you can find a description what is the lowest detectable and sortable particle size.
I think you want to separate the Exos with the specific protein from each other? Why not using magnetic beads, with the antibodies immobilized on the surface to catch them and then you can wash and analyze them (releasing maybe with slightly acidic conditions?)
This question is a bit related to a question I answered recently.
In This Paper they did it with granules, but they were much bigger in size (around 0.2 -0.4 µm)
Article Flow cytometry-assisted purification and proteomic analysis ...
Best regards and good luck!
Peter
You are welcome!
For detection of your exosomes you can also immobilize them on bigger particles. There was a question about this, which I answered recently, i try to insert a link later
After immobilization you can stain them as you wanted to do it with the fluorophore coupled antibodies and use the cytomter for detection!
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