We are using after much trial and error the following antibodies. CD25 PE from DAKO #R0808 and CD4 APC from BD #340672. Because the CD25 on these cells sometimes ranges from just above background to 10^3 this combination works. The PE 25 gives the weak 25 a boost over using FITC. On top of all this we have found the % double positives to vary depending on how aggressive the comp. settings (not to start another comp thread) the user uses. The APC works well for us on the LSR. No Comp between the two lasers. All results are clear and easily reproducible. If you are using a bead such as offered by Miltyni then you must Co-stain with a different clone. We use a PE labeled MA251 clone from Pharmingen #555432 for purity checking. Jim Houston > ---------- > From: thomasm@nshs.edu > Sent: Monday, November 11, 2002 4:43 PM > To: Cytometry Mailing List > Subject: identifying human immunoregulatory T cells > > > We have a Fellow in the lab who would like to measure > immunoregulatory CD4 t cells as part of his project. These cellos are > reported to constitutively express CD25. The literature shows them as a > dimly positive cluster, although much of the work has used bead isolation > and I'm wondering if that is modifying the receptor experssion. > > We have spent the last 2 months trying to figure out how to do this. > We have performed whole blood labeling with the thinking that the less > sample manipulation we do , the better. We have also looked at Ficolled > PBMC. We have used two different direct label antibodies [same clone, > different fluors one FITC and one APC ] . We have also tried biotin CD25 > followed by streptavidin FITC and APC. Each method has yielded > slightly > different values. We tried the enzyme amplification system without > success. > We are now ordering a different clone of CD25 McAb from a different > vendor. > > > It's unclear to me what's the best way to quantify thsi population. > Any insight on how to phenotypically identify this subpopulation would be > appreciated. > > > Thanks, > Tom > > > ************************************************************************** > ** > * > Thomas W. Mc Closkey, Ph. D. > Director of Flow Cytometry, North Shore University Hospital > Assistant Professor of Pediatrics, New York University School of Medicine > Boas Marks Biomedical Research Center, 350 Community Drive > Manhasset, Long Island, New York 11030 > ph: 516-562-4844 [office], 516-562-1135/4641 [lab] fax: 516-562-2866 > ************************************************************************** > ** > * > > > > >
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