Stimulating monocytes for IL-10
Brian A. Rabinovich (brian.rabinovich@utoronto.ca)
Mon, 29 Jul 1996 01:56:25 -0400
I am trying to develop an assay for identifying and enumerating human
monocyte IL-10 and TNF-alpha producers using flow cytometry and
intracellular cytokine staining. As of yet I have tried stimulating PBMC
isolated on a density gradient in OPT-MEM/10% FCS/antibiotics/Beta 2
mercapto for 4 hours at 37C with PMA/Iono (6/10 to 10/500 ng/ml) or LPS
(0.5 -5 ug/ml) in the presence of Brefeldin A (10 ug/ml). I am getting very
good TNF-alpha (FITC-mouse anti human TNF-alpha, Pharmingen) staining in
monocytes ( Tricolor mouse antihuman CD33, Caltag) and CD4+ cells
(Tricolor mouse anti human CD4, Caltag) but no IL-10 (PE rat anti human
IL-10, Caltag) staining. My goal is to proportionally amplify IL-10 and
TNF-alpha production in monocytes already commoitted to the synthesis of
one of these two cytokines. So far I have not found an appropriate stimulus
or perhaps there are just no IL-10 producers to stimulate from peripheral
blood. Can anyone help provide insight into a better stimulus.
Any and all insight would be appreciated greatly,
Brian Rabinovich
The Wellesley Hospital
Research Institute
University of Toronto
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