In my opinion this would be very dangerous. T cells are not the only cells to express CD4 or CD8 (monocytes & dendritic cells come to mind, respectively) so you could easily see CD4 or CD8 positive cells in your monocyte gate, which would be misleading, making you think something is positive for something it is in fact not. In theory it could be done IF two markers with the same fluoresence are exclusive - i.e., if CD4 were only expressed on T cells and another marker was only expressed on monos. In practice, this is rarely the case - NK cells can express CD3, monos can express CD4, DC's can express CD8, T cells can express CD11c, and so forth and so on. And of course there are species to species variations, so that also adds more fun to the mix. -Doug Reed -----Original Message----- From: Bunny Cotleur [mailto:bcotleur@ohio.net] Sent: Monday, January 17, 2000 7:49 AM To: cyto-inbox Subject: bleed-over from doubling up? Fellow flowers- I would like to get a consensus opinion on the following: A Fellow I work with has been "doubling up" on stain (meaning mixing lymphocyte markers, either CD4 or CD8 with monocyte/CD14 markers with the SAME fluorochrome). He feels that since you gate only on the Lymphs or mono's, there is no cross-contamination or increase signal from the wrong population. Is this valid? Does anyone else do this? Is one fluorochrome (PE/FICT/PerCP) better than another? The primary reason we would like to is because we are using human cells and barely have enough to run all the various analysis we'd like. It sounds a little too good.... -- Bunny Cotleur Cleveland Clinic Foundation Neurosciences NC30 216-444-1164 ****************************************************************** When you do something, you should burn yourself completely, like a good bonfire, leaving no trace of yourself. (Shunryu Suzuki)
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