[Cytometry] Sorting microparticles from Tcells

Howard Shapiro hms at shapirolab.com
Tue Feb 3 18:47:32 EST 2009


Monica DeLay wrote:
> First I want to say that I appreciate all of the feedback that I get when I submit questions to this list.  What a great community!
>
> For the question, I have a client that wants to sort microparticles from Tcells.  We have FACSAria II SORPs in our facility.  The smallest nozzle we have is 70 um.  My client supplied me with a very nice paper from Journal of Virology (Vol 82 No. 15 p7700 see supplementary data), where they analyzed MPs on an LSRII.  FSC and SSC are in biexponential and they use beads to set up the gate as the MPs are 0.1-0.5 um in size.  The beads were 0.1, 0.2, 0.5 and 1.0 um from Molecular Probes and were diluted 1:100,000.  They ran buffer alone as a control for background debris.  
> My client would like to stain MPs with  Annexin-FITC and CD3 (not sure what label).
>
> Does anyone have experience with sorting MPs?  I'm concerned about the nozzle size and whether the MPs will survive the sort as we usually use 70 um nozzle with 70 psi.  What about doublet discrimination?  Any other information or details to consider would be of great help.
>
>   
I haven't sorted microparticles, although I analyzed both viruses 
(published) and plasma microparticles (unpublished) in the late 1970s. 
My guess is that you have less to worry about in terms of damage with 
very small particles than with larger ones. As far as doublet 
discrimination goes, you can pretty much forget it; the way that is done 
on optical flow cytometers depends on a pulse width measurement, and the 
procedure only works for objects about the same size as the beam height 
and larger. It is impractical to make beams heights much smaller than 
about 5 um, so, although it is reasonably easy to discriminate doublets 
of particles the size of lymphocytes, it is essentially possible to do 
so when you are working with objects the size of bacteria and smaller. 
The other thing that works against you is that the distribution of 
particle sizes is something like an exponential, and doesn't have 
discrete peaks, so, even if you labeled some component of the particles 
(e.g., the membranes) using a fluorescent dye, the cytometer wouldn't be 
able to tell one big particle from two smaller ones passing through the 
apparatus close together.

-Howard



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