FYI: Monovalent cation ionophores, such as monensin, directly cause IL-1beta secretion from monocytes. This is one of the reasons I have switched to BFA. Effects of intracellular ions on interleukin-1 beta production by lipopolysaccharide-activated human monocytes. Orlinska U, Newton RC. Am J Physiol. 1992 Nov;263(5 Pt 1):C1073-80. IL-1 beta maturation: evidence that mature cytokine formation can be induced specifically by nigericin. Perregaux D, Barberia J, Lanzetti AJ, Geoghegan KF, Carty TJ, Gabel CA. J Immunol. 1992 Aug 15;149(4):1294-303. A ref that I have not read, but came up in the above search: Flow cytometric analysis of cytokine production by normal human peripheral blood dendritic cells and monocytes: comparative analysis of different stimuli, secretion-blocking agents and incubation periods. Bueno C, Almeida J, Alguero MC, Sanchez ML, Vaquero JM, Laso FJ, San Miguel JF, Escribano L, Orfao A. Cytometry. 2001 Feb 15;46(1):33-40. Calman > ---------- > From: Hodge, Greg (HAEM) > Sent: Saturday, June 2, 2001 22:48 > To: Cytometry Mailing List > Subject: RE: Problem with IL-10 > > > > > > ---------- > > 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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