I'm especially looking for technical details such as using a logarithmic scale instead of linear scale. anything else? Nozzle size etc... and what are the best antibodies for endothelial exosomes.
Mohamad - I'm imagining several people will have some thoughts on this. I would point out a couple of important issues.
First, you will need to trigger events on your cytometer with something other than forward/side scatter. Even the largest exosomes are likely to not create enough forward scatter to trigger an event, so many (if not most) will likely be lost without ever being detected. You'll need to trigger on a fluorescent marker on the exosomes to be sure and capture as many events as possible.
Secondly, for human vascular endothelium-derived exosomes, I'd consider starting with a CD31 label. One thing you'll need to do is be sure to clear all of the platelets from your samples before you start, as they also constitute a small (e.g., < 1 um) CD31+ population.
Wish I could be of more help, but it's a start. One thing you can always consider as you start to set up your instrument is to acquire a fluorescently labeled calibrating bead of about the size you imagine your exosome will be, and fully characterizing the detection and sorting of your device before going to less easily controlled clinical samples.
Good luck, and let me know how it goes.
Best, John
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Hello
What type of flow cytometer you use? If your machine is a regular one, honnestly I think you will see nothing as the limit of detection is around 500nm.
Regarding the marker for endothelial exosomes, as exosomes of other cell types, I think TSG 101, CD9, CD63, CD81 and tetraspanin are good markers for most of exosomes (if I remember well i think CD9 is not always expressed in exosomes, so be carreful).
Regarding technical details. Always acquire at low (due to the small size of exosomes you have a high risk of swarm detection), add controls to distinguish microparticles (produced by plasma membrane buddings) and exosomes but also protein aggregates.
To have an idea you can use calibrating beads (silice ones are better than silicone ones) but in this size range, you will obtain only an approximative size (since you are under 488 the wave length of the blue laser used to detect the FSC) if you have a very powerful cytometer. And as John said, use the fluorescence and not the FSC SSC parameters to trigger events on your cytometer.
Good luck
Matthieu
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