inconsistent results in monocytes

From: Maciej Simm (simmmmer@yahoo.com)
Date: Mon Nov 13 2000 - 13:05:41 EST


I'm stumped:

I activated whole blood with LPS for 4hrs, in presence of BFA, with
all of the pertinent controls. I ran the assay 3 times, it only
worked once. By "worked" I mean I detected IL-10.

Is there some other way to get a "high" control for IL-10? Any human
Leukocyte will do.

I am using a Rat-anti-human IL-10, IgG2a, R-PE conjugated.

Also, what would be the range of cells positive for IL-10 in this
setup?

I'm using the BD protocol found at
http://www.bdfacs.com/literature/brochures/cytokine.pdf

any help would be appreciated,
Maciej

PS. other cytokines run in parallel worked - IL-1b and TNFalpha. So
it's not the permeablization step as far as I can tell, nor any of
the wash steps or experimental setup.

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