Characterisation of IL-12-producing cells. Kinetics of the IL-12 response to CpG-2006 during the first 48 h was followed by ELISA. Mean ± SEM IL-12 secretion (CpG-2006 - Ctl-ODN) is shown for each time point. Means ± SEM were calculated from three independent experiments (three different neonates) giving similar results (A). Neonatal MLN cells were cultured in vitro with 1 μM Ctl-ODN (hatched bars) or 1 μM CpG-2006 (black bars) for 25 h, with brefeldin A added for the last 5 h. IL-12 intracellular staining was combined with labelling of CD11b, CD14, MHC class II and CD205. Values reported are means ± SEM from three independent experiments (three different neonates) giving similar results (B). Neonatal CD14+ MLN cells were observed by transmission electron microscopy. The scale bar on the representative picture indicates 2 μm (C). Neonatal (n = 11, closed bars) and adult (n = 6, open bars) total MLN cells and sorted CD14- cells and CD14+ cells (n = 6 for neonates and n = 5 for adults) were stimulated for 48 h with 1 μM CpG-2006 or 1 μM Ctl-ODN and ELISA was used to assay secreted IL-12 (CpG-2006 - Ctl-ODN) (D). Mean ± SEM of CD14+ cell proportions in 6- to 14-day-old neonatal (n = 27, closed bar) and adult (n = 13, open bar) total MLN cells were assessed by single staining and flow cytometry (E).* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.