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Date: Thu, 12 Oct 2006 09:45:57 +0200 (CEST)
From: Karen Hensen <karen.hensen@virgajesse.be>
To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
Subject: Re: non-functional T regs after FACS sort on ARIA
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Hello Anja

We have experience sorting Tregs and they kept their suppressive activity.The results are
published in  J Neurosci Res. 2006 Jun;83(8):1432-46. No special sorting conditions
required. We sorted on a FacsAria under high pressure conditions (20 00-30 000
events/sec)  in tubes containing RPMI medium with 30%FCS. It's important to wash cells
after sorting because the sheet fluid has negative effects on viability of cells.

Beste regards

Karen





Karen Hensen, PhD 
Laboratory of Experimental Hematology 
Virga Jesse Hospital 
Stadsomvaart 11 
3500 Hasselt 
Belgium 
Phone +32-11-309700 
Fax +32-11-309750

--- Oorspronkelijk bericht ---
> Dear all.
> 
> Maybe someone can help me with the following scenario: 
> 
> I recently sorted CD25 high CD4 T cells (potential T regs that
>  do stain positive for
> FoxP3) on a FACS Aria. I tested the cells for suppressor
>  activity in a well established
> antigen-induced proliferation assay in our lab (using autologous
>  whole PBMCs as
> responders), but couldn't detect any suppressor activity. 
> 
> We usually use MACS separation to isolate out T regs, but a
>  colleague of mine also tried
> FACS sorting once with the same result: The T regs had no
>  suppressor function (while they
> do after the MACS sort).
> 
> I am planning to repeat the sort next week (I can't do MACS
>  as I want to distinguis
> several different populations), but would hate the experiment
>  to be wasted again because
> I missed some crucial points.
> 
> Does anyone here have experience with sorting CD25high CD4 T
>  cells for subsequent
> functional assays and can tell me, whether there is anything
>  I have to be especially
> careful with to maintain suppressor function, such as low vs.
>  high pressure sorting (we
> sorted at 10.000 events/second), the media that you sort into
>  (I used PBS/EDTA with 10%
> autologous serum to keep cells from aggregating but still
>  happy) or anything else I can't
> think of right now?
> 
> Any suggestions are much appreciated!
> 
> Cheers, Anja
> -- 
> _______________________________________________
> Anja Scholzen
> PhD Student
> 
> Burnet Institute at Austin
> Vaccine Development and Infectious Diseases Unit
> Studley Road, Heidelberg 3084
> VIC, Australia
> Tel.:0061 3 9287 0673
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