PKC inhibition of intracellular cytokine production in whole blood

Patrizia luppip+ at pitt.edu
Wed Mar 6 22:29:17 EST 2002



Dear flow cytometry experts:

I am trying to study the effect of protein kinase C inhibition on
intracellular cytokine production of PMA/ionomycin-stimulated human
monocytes and neutrophils in whole blood. I am particularly interested
in phagocyte production of IL-1b, TNF-a, IL-6 and IL-8.

As a pan-specific protein kinase C inhibitor I have tried Calphostin C
at a final concentration of 50nM in whole blood (with a pre-incubation
of 1 hour under  light before adding PMA/ionomycin and brefeldin A). The
problem I have is that addition of Calphostin C to whole blood of
different donors tested at the same time is giving inconsistent results,
i.e., in one donor is decreasing the percentage of cytokine positive
cells while in another the percentage of the same cells positive for the
same cytokine are increased or show no change. I have tried several
times and thought of possible interpretations but with no success. I
have been performing intracellular cytokine staining routinely since
three years and therefore I am very confident of the technique. I have
searched for literature on the topic but couldn't find anyone who had
reported investigations on PKC inhibition of intracellular cytokines in
whole blood.

I would greatly appreciate any suggestion or insight or indication of
literature that could help me solving this problem.

Thank you very much in advance for your time and help,

Patrizia Luppi, M.D.
Div. Immunogenetics, Dept. of Pediatrics
University of Pittsburgh Schhol of Medicine, USA




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