Re: gating CD45RO+/RA+ Lymphocytes

From: Margaret Tropea (mtropea@nih.gov)
Date: Wed Aug 30 2000 - 07:44:16 EST


I think that this is best handled using three color flow.  The CD3 should
be in FL3 and CD3+ vs ssc should be used for the gate.  The CD45RA/CD45RO
tube should also contain the FL3 CD3 label.  This insures that you look at
all of the T cells and gives you the percentage of single positives, double
positives and double negatives in the total CD3 positive population.  I
usually collect 2000 CD3 positive events.  I agree with Mario Roederer
comments that if you want to enumerate memory vs naive cell populations
CD45RA vs CD62L (this is the only one I have experience with) or his other
suggestions are better choices for enumerating memory vs naive T cells.

Margaret



>Monica,
>
>I would create a dotplot of ssc vs. CD3 and gate on the region low on
>SSC axis and bright for CD3, to get all my T cells in the picture.
>
>Then I would create a dotplot of CD45RA/CD45RO. I don't use the same
>colors as you do but RO+ cells are just above the isotype whereas RA+
>are a bit brighter than isotype.
>
>It would help if you could post some pictures of the dotplots you are
>working with to see what you mean by difficult gating.
>
>Regards and good luck,
>
>maciej
>
>
>
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>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Margaret M. Tropea
Critical Care Medicine Department
National Institutes of Health
(ph) 301-496-7752
(fax) 301-480-3389
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>



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