Kathy, CD4 is downregulated in a matter of minutes through nearly all stimulations (PKC-dependent phosphorylation of a cytoplasmic amino acid signals the molecule to be endocytosed). We have overcome this problem by simply pre-labelling cells with antibodies to CD4. When the CD4 molecule is internalized, so is the antibody and its fluorochrome. The major problem is the acidification and proteolysis that occurs during endocytosis, which kills many fluorochromes (FITC, PE, Cy5PE, APC, PerCP...). HOWEVER, if you include monensin from the beginning (monensin also inhibits Golgi export, and is a very cheap alternative to Brefeldin A), then the acidification and proteolysis does not occur--thereby rendering CD4 cells fully fluorescent! Note we use this protocol for 6 hour stimulations, not 24. I don't know if it will work for 24 hours... However, the alternative is to include the anti-CD4 antibody with the intracellular stains--after all, the CD4 is internalized, and should be labeled by IC staining. This presumes that it hasn't been fully degraded (and not resynthesized) during the 24 hour period, but it's definitely worth a try. Note that you MUST re-titre your anti-CD4 antibody if you want to use it for intracellular staining--the titre will be different than that for surface staining. Finally, we haven't yet published this information, but I've been presenting it at meetings for a couple of years. We are putting two papers out in the next few months that will have it; in the meantime, you can just reference personal communication if you wish. mr >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
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