Fig. 8.

Prolonged LPS-free pre-incubation of monocytes with HNSCC Cal27 cells enhanced the suppression of LPS-induced TNF-alpha production. Donor 1 monocytes were cultured alone or co-cultured with Cal27 for 1, 2 or 3 days before stimulating with 200 ng/ml LPS for 6 h in the presence of Brefeldin A, labeled with anti-CD11c-Cy-5-PE antibodies, briefly fixed, permeabilized and stained with anti-TNF-alpha-PE antibodies or negative control. In parallel wells, each LPS stimulation continued for 24 h and supernatants were evaluated for secreted TNF-alpha by ELISA. a Histograms of intracellular TNF-alpha in monocytes: dotted line = negative control; solid thick line = TNF signal without LPS; shaded histogram = TNF signal with LPS stimulation. MFI of the TNF signals are shown in the upper right corner of each plot, in the absence of LPS (top number) and in the presence of LPS (bottom number). b Relative values of intracellular and secreted TNF-alpha in LPS-stimulated MO-Cal27 co-cultures as percent of control LPS-stimulated monocytes cultured alone. The results are representative of three independent experiments.