The BD clone works fine in our hands when used at the same time as the anti-cytokine mAbs and as a PE or APC conjugate. The CD69 is intracellular, due to the use of brefeldin A; therefore if using BFA you must stain after permeabilization. Given that TNF and IFN-g are pretty bright, you should have no problem getting a free channel for use of CD69 PE or APC. >From your email I'm not sure if you have tried looking at intracellular CD69 in PMA/ionomycin activated, fixed and permeabilized cells. Would start with that as a positive control. Also, how long are you activating your cells for? 6 hours is enough to see CD69. Calman > _______________________ > Calman Prussin > Laboratory of Allergic Diseases > NIAID/ National Institutes of Health > ---------- > From: Michal Abel > Sent: Monday, September 2, 2002 17:39 > To: Cytometry Mailing List > Subject: missing CD69 > > > 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 > >
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