Hi Michal, I suppose that if you are doing ICCS then you must be using brefeldin A or monensin. I think your problem is here as CD69 may also be inhibited by these reagents from being transproted to the cell surface. I am not sure how to get around this problem except for doing ICCS for CD69 also. Hope this helps Regards adn best of luck Rana --- Michal Abel <abelm@necker.fr> wrote: > > Dear experts in flow cytometry, > > I am performing an intracellular staining of > lymphocytes for INF and TNF. These > markers work very well after PMA or peptide > stimulation. My problem is that all > INF or TNF positives cells are CD69 neg. > I have tried CD69 from Immutech and from BD. Both > works well on non-fixed > cells. Unfortunately, I cannot visualize CD69+ after > fixation. I have try to > fix with perm&fix kit (Caltag), BD fixation sol. and > classical > saponine/formaldehyde method. I have also tried to > stain with CD69 before and > after fixation. I have always had all CD69 at 101log > (same for PMA > and peptide activation and non activated control > cells). > Can someone tell me what I am doing wrong, please ? > > Thank you for all your help and any ideas ! > > Michal > > -- > Michal Abel, MD, PhD > Laboratory of Clinical Immunology > INSERM U25 > Necker Hospital, 161 Rue de Sèvres. Paris > tel: 33 1 42 19 28 87 > fax: 33 1 44 49 53 74 __________________________________________________ Do You Yahoo!? Yahoo! Finance - Get real-time stock quotes http://finance.yahoo.com
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