Kathy, Dr. O'Gorman in Chicago has a nice technique for accomplishing this. He stains with CD3 and CD8, then gates the CD3+, CD8- events and assumes these to be the cells which would express CD4 (has they not been overstimulated). I believe he described this in a paper concerning a CD40L assay he developed. Keith Bahjat Graduate Assistant University of Florida kbahjat@ufl.edu Kathleen Schell wrote: > Dear Flowers, > We have an investigator who is studying cytokine production in > response to mitogenic stimulus. We observe a down regulation of the CD4 > molecule ( in surface staining) by 24 hours that renders this population > unidentifiable. They are looking at intracellular cytokines, and surface > molecules are stained first, cells are fixed, permeabilzed, and then > stained for cytokines. Has anyone seen this? We have repeatedly seen a > decrease in the fluorescence of CD3 after PHA stimulation, but never to the > point where the population can no longer be seen--this has been reported in > the literature. Is there another way to identify the CD4 population using > this kind of staining protocol? Cells not stimulated have good surface > expression of CD4. > Thanks in advance. > Kathy > > -------------------------------------------------------------- > Kathy Schell > Supervisor, UWCCC Flow Cytometry Facility > 600 Highland Ave. K4/535 > Madison, WI 53792 > Voice: 608-263-0313 > e-mail: kschell@facstaff.wisc.edu
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