Monica (and anyone else doing T cell subsetting!), unfortunately, you CANNOT accurately (not even close) identify naive and memory T cells using that combination. First of all, the expression of CD45RA on CD8 T cells is different than that on CD4--you need to use a different gate for CD45RA on CD8 cells. In fact, the "CD45RA-" cells among the CD8 compartment actually express CD45RA, and many almost as much as CD4 naive T cells. But another problem is that there are significant fractions of CD45RA+ (and CD45RO-) cells that are memory. These can only be distinguished by using another marker, such as CD62L, CD27, or CD11a. (For concrete examples of this, see two papers in JCI, volumen 95, pages 2054-60 and 2061-6). There is no point in using CD45RA together with CD45RO to measure resting T cell subsets. I've been trying to get this point across to the immunophenotyping community for 5 years! CD45RA and CD45RO provide essentially the same information; it is slightly preferable to use CD45RA only because it is brighter than RO; however, either one works. We have a paper in press in Nature Medicine, should be out in October or November, which provides some very detailed information about the use of various markers to identify naive & memory subsets in humans. The bottom line is that if you are doing 3-color, you should use two panels (one for CD4 and one for CD8), which are CD4 (or CD8) plus CD45RA (or CD45RO) plus CD62L (or CD27 or CD11a). mr (PS, there is a case for using both RA and RO together: and that is to identify T cell blasts or activated T cells; as Picker showed, early after activation, both of these markers are co-expressed AT HIGH LEVELS--not the dull co-expression that can be found on some memory subsets). At 9:35 PM +0200 8/22/00, Monica Matas 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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