Dear colleagues, to my question regarding the best stimulus, time point and antibody to detect human IL10 intracellularly I got the following responses: > The only stimulus we have gotten to work for IL-10 in fresh T cells is PMA/iono at 6 hours. We have had good results with the 9D7 clone, preferably conjugated to PE or APC. The frequency of IL-10 pos CD4 T cells is typically <<1%. > at a recent meeting A. Radbruch showed that the best moment to stain cells for IL-10 production is at 10 hours after the stimulus. The data he showed, if I remember correctly, was obtained using the Miltenyi kit for the identification of VIABLE cytokine-secreting cells (works very nicely). Apparently ionomycin and PMA are not the best stimulus, and PHA is used. We routinely use both the Miltenyi system and the standard intracellular staining, with good results. > We've been staining intracellularly IL10 according to BD Fix & Perm protocol; using PE- and APC-labeled antibodies. This was done in CD4+ and CD8+ T cells. > 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. We also use the Miltenyi kit regularly and these give very good and repreducable results http://home.wanadoo.nl/flowcytometry/MB100401_files/v3_document.htm ), but we would like to stain intracellularly to be able to combine it with other cytokines. When I got a working protocol I will include that in my website http://home.wanadoo.nl/flowcytometry/ ). Thanks to everybody that responded! Kind regards, Joost Schuitemaker
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