PBMCs obtained from peripheral blood samples of healthy volunteers were cultured in RPMI containing 10% FCS, 4 ng/ml IL-2, 50 ng/ml IL-4 and 50 ng/ml GM-CSF for 72 h, followed by stimulation with 10 ng/ml PMA and 100 ng/ml LPS for 16h. Cells were then treated with 10 μg/ml of cycloheximide, lysed and post-nuclear supernatants were centrifuged at 40,000 rpm for 2 h 15 min through 12 ml linear 20-47% (w/v) sucrose gradients containing 20 mM Tris-HCl pH 8.0, 5 mM MgCl2, 140 mM KCl. EDTA control gradients contained 20 mM Tris-HCl pH 8.0, 10 mM EDTA, 140 mM KCl. 1 ml fractions were collected and absorbance at 260 nm was used to generate chromatograms of sedimentation profiles. RNA was extracted from the fractions and cDNA was analysed by qRT-PCR.
