When you note "mitogenic stimulus" do you mean PMA/ionomycin? PMA down regulates surface CD4 expression at concentrations from 1 to 20 ng/ml and as early as 4 hours. By 24 hours there is little if any CD4 to be detected. There are several "tricks" to this problem, none of which is completely satisfying. 1. Stain intracellular CD4. My experience is that after PMA activation, intracellular CD4 stains far better than extracellular CD4. My guess is that CD4 is modulated off the cell surface. Try staining both CD4 and cytokines simultaneously. 2. 4-6 hours activation. Why are you activating for 24 h? This is not an ELISA. Cytokine generally peaks at 6-18h. The CD4 staining will get worse as time goes on. 3. Try staining for CD3+, CD8- cells. Yeah, I don't like it much either, but it works and has been used in a number of papers. 4. Titer your CD4 mAb. In my experience, CD4 staining is harder than the cytokine staining! Some CD4 clones work better than others in these situations. Does PHA give anyone much signal in regards to intracellular cytokines? Calman > _______________________ > Calman Prussin > Laboratory of Allergic Diseases > NIAID/ National Institutes of Health > > ---------- > From: Kathleen Schell > Sent: Monday, February 1, 1999 15:29 > To: Cytometry Mailing List > Subject: modulation of CD4 > > > 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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