[Cytometry] microvesicle analysis

Joanne Lannigan jl7fj at virginia.edu
Thu Feb 6 12:20:51 EST 2014


Hi Uttara:
At the present time it is unlikely that you will be able accurately identify individual microvesicles that are less than 0.500u, this would exclude exosomes which are generally 50-100nm and are the vesicles most like to contain RNA. Please do not try and size microvesicles using beads as this will give you very inaccurate results due to the differences in their refractive indices. The best way to prove your level of detection sensitivity in terms of size on your flow cytometer is to use fluorescently labeled liposomes of various sizes as they better represent the type of microparticles you are trying to detect. Mostly what is detected by flow cytometry when you trigger on scatter are swarms of microvesicles, i.e. a single triggered event is caused by multiple MVs in the laser simultaneously. If you dilute your samples and your signal disappears then you are not seeing individual MVs. See  Van Der Pol et al J. of Thromb. and Haemostasis, 10:919-930 (2012) You have a better chance of detecting small particles above background using a fluorescence trigger instead of a scatter trigger. See Nolan et al Cytometry (A) 83A:301-305, 2013. 
Best regards,
Joanne

Joanne Lannigan, MS
Director, Flow Cytometry Core
University of Virginia 
School of Medicine
1300 Jefferson Park Ave.
Jordan Hall, Rm 2011A
Charlottesville, VA 22908-0734
flowcytometry at virginia.edu
office: 434-924-0274
lab: 434-243-2711

On Jan 28, 2014, at 6:32 AM, Uttara Chakraborty wrote:

> Dear Flow Community,
> I have some queries regarding microvesicle analyses by flow.
> 
> Is it possible to observe distinct scatter properties of microvesicles in FACS Aria III. If so, what are the parameters that should be taken care of and what size range will they fall into.
> Do they have any RNA content when they are shed off.
> Is it possible to use RNA specific dyes to exclude exosomes from my population of interest in the same size range.
> Eagerly awaiting your suggestions and advice.
> Thanks in advance.
> Uttara
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