Try using 6 hour PMA/ionomycin activated human PBMC as a positive control. If that works you should try cyno PBMC. you may wish to use a PE or APC conjugate if you want greater brightness. > ---------- > From: Reed, Doug S Dr USAMRIID > Sent: Wednesday, January 30, 2002 16:19 > To: Cytometry Mailing List > Subject: CD69 staining > > I've got a question for the list regarding CD69 expression, or rather the > inability on our part to detect it. > > We've been staining primate (cyno) PBMC, looking at the host response to > viral infection. Everything looks great, but nowhere do we see any CD69 > expression. Compared to negative controls we see a slight - very slight, > by my eyes - shift, nothing I'd want to write home about. Back a few years > ago when I was knee-deep in mice in Connecticut we tried it with mice and > also saw very poor staining - again, hardly anything I was impressed with > at the time. I look in the literature, however, and I see beautiful CD69 > staining. I've left this alone for a while, not really a front-burner of > an issue, but it has lurked there out of sight, waiting for me to pick it > up again. > > Last night I sat down and read a paper in JI from Louis Picker's group in > Oregon, looking at memory cells in the rhesus macaque. Looking at the > methods it clearly states that for intracellular cytokine staining the > cells are stained for surface antigens, fixed & permeablized, and then > stained with CD69 and cytokine mAbs. So I'm wondering - is that where I've > been going wrong? Any thoughts? I tried searching the archives but most of > the messages pulled up by CD69 deal with it's relationship with > proliferation. > > Thanks! > > Doug > > Douglas S. Reed, Ph.D. > Microbiologist > Respiratory & Mucosal Immunity > Department of Aerobiology & Product Evaluation > Division of Toxinology & Aerobiology > U.S. Army Medical Research Institute of Infectious Diseases > 1425 Porter Street, Fort Detrick > Frederick, MD 21702-5011 > 301-619-6728 > 301-619-6911 fax > doug.reed@det.amedd.army.mil > >
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