> ---------- > From: Monica Matas > Sent: Wednesday, 23 August 2000 5:05 > To: Cytometry Mailing List > Subject: gating CD45RO+/RA+ Lymphocytes > > Monica, > > A reference on CD45RA/RO by S. Hodge et al "Surface activation markers > of T lymphocytes.....", Clin Exp Immunol 1998; 113:33-38 may help. It is > important to titre the CD45RO PE and CD45 RA correctly. > We used BD reagents but I think the same would apply to other reagents. > Untitred, the cells quite often sit beyond the neg control. Sometimes the > neg control is useless and a coloured contour plot may help identifying > the 3 populations. If you stimulate cord blood or neonatal pbmcs > (essentially RO neg) with 10 ug/ml PHA at about day 3 the dual staining > occurs. At around day 5 most cells are 45RO pos. Can serve as good > controls until you get the feel for quad marker setting. In the above > article, we found that the dual pos RA/RO was easily identifyable in the > early stages of neonatal infection. > Hope this helps. > > Regards, > > Greg Hodge, PhD > Flow Lab/ Haematology Department, > Women's and Children's Hospital > North Adelaide, > South Australia 5006 > ph: 08 82046522 > fax: 08 82046043 > email: hodgeg@mail.wch.sa.gov.au > > > -------------------------------------------------------------------------- > --------------------- > > > > > On 23/8, Monica wrote: > > I am a student of nutrition science at the university of Bonn (Germany) > and > I'm working on a study of immunophenotyping T-lymphocytes by three-color > Flow cytometry. > I want to get the percentages of the naive T-cells (CD45RA+) and memory > T-cells (CD45RO+) in the blood samples. Therefore I used the > antibodycombination of CD45RA-FITC, CD45RO-PE and CD3PerCP. > I'm experiencing difficulties by setting the gates in the dot plots. I > don't > know where to set the gates for separating the two populations of cells > (naive and memory T-cells).I don´t know what criteria I have to use to > separete this two subpopulations. > (I'm using the program WinMDI 2.7) > I know there are some subpopulations between the naive and memory T-cells > like double positive T-cells (CD45RA+CD45RO+) and this makes it very > difficult to set the gates. > > I will be very obliged if you could tell me something about it or if you > could tell me about related literature. > > Thank you very much in advanced. > Monica Matas Nobis > > --------------------- > Mónica Matas Nobis > mailto:M.Matas@gmx.de >
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