MLR's with Flow

Andy Heath a.w.heath at sheffield.ac.uk
Tue Nov 22 05:24:23 EST 2005


Hi Don,

I don't think you even have to purify the T cells first. You can resolve
them at the end by labelling with anti-CD3 or CD4 and gate just on those
cells, and then look at dilution out of your CFSE analogue. We're doing
something similar, albeit using CFSE itself, and it works fine.

Andy



-- 
Andy Heath

Dr A.Heath
Reader in Immunology
Infection and Immunity
F Floor
University of Sheffield Medical School
Beech Hill Rd
Sheffield S10 2RX







> Hi Don,
> 
> Have you tried going for a CFSE-dilution type experiment? Assuming you first
> purify the T-cells, say on a MACS, I'd stain them with a FACSarray
> compatible CFSE analogue, mix with unlabeled APC's and try to observe
> downward shifts in fluorescence intensity.
> 
> CFSE won't work on the Array, but I've got two non-overlapping spectra dyes
> working already and a third one waiting to be tested. Mail me directly if
> you want the gory details.
> 
> Guy
> 
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> 
> -----Original Message-----
> From: Wigington, Don [mailto:Don.Wigington at genzyme.com]
> Sent: Friday, November 18, 2005 12:12 AM
> To: cyto-inbox
> Subject: MLR's with Flow
> 
> 
> Dear Flowers
> 
> Here is a pretty simple question. (But I bet not a lot of simple answers.)
> 
> Has anyone ever tried to use flow cytometry for analyzing mixed leukocyte
> reactions? I am trying to study the proliferation of responder T cells rate
> mixed with drug treated APC's. We can't use radioactivity here so I was
> going to try a colorimetric approach (cell titer 96;Promega) but the
> sensitivity is not great enough. I am a bit worried that I will not have
> enough cells for flow but I do have a BD FACSArray so I can do it in 96-well
> plates. Any info would be appreciated very much.
> 
> Don
> 
> -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-
> Don Wigington
> Manager, Preclinical Laboratory
> Genzyme Corporation
> 1600 Aspen Commons
> Middleton WI 53562
> 608-662-7866	fax 608-662-7870
> 
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