Doug, I'm not sure if the following addresses your problem or is is of any help to you, but use of CD69 as quality control for surface activation and intracellular staining in human whole blood is well decribed in the BD application note: detecting intracellular cytokines in activated lymphocytes (www.bdbiosciences.com). As I understand it, it depends on whether you are using CD69 as an activation control (surface expression) or for intracellular staining (permeabilization control). The former demands there be no BFA during incubation, to permit surface CD69 expression, and you can directly stain surface CD69 (should be >90% pos). The latter situation is as you described below: You need an anti-secretory agent like BFA, then Ab to any other surface ags are 1st stained and PFA fixed, followed by permeabolisation, anti-cytokine and anti-CD69 to detect intracellular (CD69 should exceed 90%) staining. I'd be interested to see what others, particularly those working with macaques, have to say. Regards & good luck. Stan > From: "Reed, Doug S Dr USAMRIID" <Doug.Reed@DET.AMEDD.ARMY.MIL> > To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> > Subject: CD69 staining > Date: Wed, 30 Jan 2002 16:19:18 -0500 > 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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