Expression of COX-2 protein in trigeminal ganglia cells after IL-1β stimulation.

<p>COX-2 protein in cell culture homogenates was analyzed 6 hours after stimulation with IL-1β using Western blot (n = 3). A representative image is shown in panel <b>A</b>. Cell lysate of IL-1β treated TGC and the positive control (IFNy/LPS treated macrophages) showed a clear band at 70 kDa corresponding to COX-2 protein. Vehicle stimulation resulted in a faint COX-2 expression. The expression of COX-2 protein in cultured trigeminal ganglia cells exposed 6 hours to vehicle (10 ng/ml, upper panel B1-B3/B7-B9) or IL-1β (0.1 M PBS, lower panel B4-B9/B10-B12) is shown in fluorescent micrographs in panel <b>B</b>. Cells were stained with a mouse β-tubulin III antibody, indicative of neuronal cells (B1 and B4) or a mouse GFAP antibody, indicative of glial cells (B7 and B10), and a rabbit COX-2 antibody (B2, B5, B8, B11). The β-tubulin III and the GFAP antibodies were recognized by an Alexa Fluor 488 labeled secondary donkey anti-mouse antibody (green) and the COX-2 antibody was recognized by an Alexa Fluor 594 labeled secondary donkey anti-rabbit antibody (red). Double stained cells appear orange in B3, B6, B9 and B12. IL-1β caused a clear upregulation of COX-2 in neuronal and glial cells (lower panel) whereas a faint COX-2 expression could also be observed in control experiments (upper panel). The strongest induction of COX-2 was seen in bigger glial cells (40-100 µm) and neuronal cells.