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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