Mucin-mediated COX-2 induction and PGE₂ production

 Prepare culture medium of colon cancer cells.

 Fractionate the culture medium by gel filtration on a Sepharose 6B.

 Add perchloric acid to the mucin fraction to a final concentration of 0.5 M.

 Centrifuge at 10,000 rpm for 30 min at 4°C ( Hitachi, RPR20-2 rotor ) and discard the precipitates.

 Neutralize the supernatant with 1 M Tris-HCl, pH 8.0 and dialyse against dist. water.

 Reduce the sample by incubation in 0.1M Tris-HCl, pH 8.0, 2mM EDTA, 6 M guanidine-HCl, and 0.1 M dithiothreitol for 2 h at room temperature.

 Alkylate the sample by treatment with 0.3 M iodoacetamide for 2 h in the dark.

 Add guanidine-HCl to a final concentration of 4 M and cesium chloride to a final density of 1.25, and centrifuge at 36,000 rpm for 72 h at 10°C ( Hitachi, P40ST rotor ).

 Prepare peripheral blood mononuclear cells ( PBMC ) from human peripheral blood on Ficoll-Isopque.

 Isolate monocytes from PBMC by using anti-CD11b antibody coated magnetic beads.

 Treat the monocytes with mucins ( 0.1–1 μg/mL ) in RPMI1640 medium supplemented with 10% FCS for 24 h.

 Determine the PGE₂ level in the culture medium by ELISA.

 Treat the monocytes with mucins ( 0.1–1μg/mL ) in RPMI1640 medium supplemented with 10% FCS for 4 h.

 Prepare total RNA from the monocytes.

 Estimation of COX-2 mRNA level by real time PCR.