You can detect less than 1% IL-10 producing cells in lymphocyte population after 6 hours stimulation with PMA plus ionomycin; if you stimulate purified human CD4 T cells with immobilized anti-CD3 mAb plus soluble anti-CD28 mAb for 24-48 hours, much higher % of IL-10 positive cells can be detected. Adding monensin or brefeldin A in the culture system for the last 6 hours stimulation is important for the intracellular staining of cytokines. Xifeng Yang, M.D. eBioscience http://www.ebioscience.com ----- Original Message ----- From: Joost Schuitemaker To: Cytometry Mailing List Sent: Thursday, May 10, 2001 3:29 AM Subject: Intracellular staining of IL10 Dear friends, For a few years now intracellular staining of cytokines is performed almost routinely in some labs (including ours), but there are still some cytokines that give difficulties. For instance TGF's, IL18 and IL10. And especially this last one is more and more investigated by many people. Detection (intracellularly) of IL10 production by monocytes after stimulation with LPS or CD40L is no problem. But what about lymphocytes? We would like to look at this cytokine in HUMAN T cells. And when possible in combination with other cytokines. Has anyone ideas concerning timepoint, kind of stimulus and antibody to use? Litt. is not conclusive and detection in ELISA is much earlier than seeing 0.0X % IL10 positive cells (with the same stimulation) in intracellular staining. Can someone help us? Kind regards, Joost Schuitemaker ______________________________________________________________ J.H.N. Schuitemaker Senior Research Technician Dept. Cell Biology & Histology Cellular Immunology Group AMC, University of Amsterdam Meibergdreef 9, L3-351 PObox 22700 1105 AZ Amsterdam 1100 DE Amsterdam The Netherlands Interested? VISIT: (intranet) http://m3.amc.uva.nl/celbiologie (internet) http://www.amc.uva.nl/ and (internet) http://home.wanadoo.nl/flowcytometry/ Telephone: +31 (0)20 5664960 Fax: +31 (0)20 6974156 E-mail (privé): J.H.N.Schuitemaker@consunet.nl
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