> ---------- > From: philipp > Sent: Thursday, 31 May 2001 18:53 > To: Cytometry Mailing List > Subject: Problem with IL-10 > > Philip, > > I regularly stain for IL-10 in LPS stimulated whole blood monocytes > at 24 h. I use 100ng but 1ug/ml works also. IL-10 like IL-12 is produced > later than other cytokines in stimulated monocytes. Some workers have > found that priming with 100 IU IFNg improves IL-10 production but LPS > alone will give good results (5-15%). PMA stimulation will result in loss > of CD14 as will high doses of LPS. I regularly add 100 uL 20mM EDTA and 10 > uL human Ig (60 mg/ml) and vortex tubes to detatch monocytes and block Fc > receptors at end of culture period. I have found 10 mg/ml BA to give > consistently greater percentages of IL-10 pos monocytes but Monensin works > OK. For controls I stain cells from same tube for IL-2 or IFNg, cytokines > not made by monocytes, to set quad markers. Hope this helps. > > Greg Hodge PhD. > -------------------------------------------------------------------------- > -------- > > > > Hi > > I am measuring intracellular Cytokines in Monocytes, but I am getting > bad results with IL-10, all other Cytokines work great with my assay. > > I get about 2-5 % positve Cells after 6h Stimulation with LPS (10nM), > but even these results are very unstable. With PMA and Ionomycin the > results vary much more than with LPS. Mostly I can not detect IL-10 at > all. So I got some questions about this matter. > > 1. Which Stimulus works best to get IL-10 ? and which Concentration you > use for Stimulation ? > > 2. Which point of time is the best to measure IL-10 ? > > 3. How much positive Cells I have to expect at that point of time and > with which Stimulus ? > > 3. Which Golgi-Inhibitor is the best to measure it ? Monensin or > Brefelidin A ? what concentrations you use? > > 4. Is it possible that i loose my monocytes, which are IL-10 postive > through cell adherence in my Tubes? > > > Thanks in advance > > Philipp > > > > > Philipp K. Röntgen > > Department of Thoracic Surgery MLU-Halle > Heinrich Damarow Str.1 > 06120 Halle > Germany >
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