> I want to get the percentages of the naive T-cells (CD45RA+) and memory T-cells (CD45RO+) in the blood samples. < An interesting alternative for phenotypic identification of naive T cells has been recently proposed by Fagnoni et al. (Blood, 2000, 95:2860-68) based on Fas/CD95 absence of expression. One advantage is that the method would require only one color rather than 2 for CD45RA/CD62L. I am not sure whether the limit between the so-called CD95 negative cells and the positives will be that easy to place using isotypic control since in our experience with adult blood samples they express low but distinguishable levels of about 500 copies of Fas (whereas the so-called CD95 positive cells express about 10,000 copies ) . Using quantitative flow cytometry, a standardized limit between Fas-low ( naive ) T cells and Fas-high ( activated and memory ) could be set at about 3,000 CD95 molecules per cell. In any case, the alternative of using CD95 should be taken into consideration. Philippe Poncelet, PhD BioCytex , France www.biocytex.fr "Monica Matas" <M.Matas@gmx.de> le 22/08/2000 20:35:02 Veuillez répondre à M.Matas@gmx.de Pour : Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> cc : (ccc : MailingList/general/Biocytex) Objet : gating CD45RO+/RA+ Lymphocytes 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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