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
CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web
http://www.cyto.purdue.edu
, EMAIL
cdrom3@flowcyt.cyto.purdue.edu
CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web
http://www.cyto.purdue.edu
, EMAIL
cdrom3@flowcyt.cyto.purdue.edu
