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 > > > >===== >`---------------------------------------------` >| Maciej S. Simm | 525 E 68th Street | >| Research Technician | Room N-805 | >| Cornell Medical Center | Tel. 212.746.3428 | >`---------------------------------------------` >|www.cd4cd8.com | 917-734-6280 | AIM - XsimmX | >`---------------------------------------------` > >__________________________________________________ >Do You Yahoo!? >Yahoo! Mail - Free email you can access from anywhere! >http://mail.yahoo.com/ >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> Margaret M. Tropea Critical Care Medicine Department National Institutes of Health (ph) 301-496-7752 (fax) 301-480-3389 >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
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