R. D’Elia and K. J. Else Parasite Immunology Figure 1 Bone marrow macrophages (BMMΦ) stimulated with parasite speciﬁc E/S show no consistent change in co-stimulatory cell surface markers compared to unstimulated levels. Bone marrow was isolated and pooled from 3–5 naïve C57BL/6 mice and cultured into BMMΦ. The BMMΦ were incubated for 48 h, in vitro with medium alone – unstimulated (U), 50 μg/mL E isolate E/S (E), 50 μg/mL J isolate E/S (J), 50 μg/mL S isolate E/S (S) or 100 ng Lipopolysaccharide (LPS). The cells were analysed for cell-speciﬁc markers F4/80, CD11b, co-stimulatory markers CD80 and CD86 and antigen recognition marker MHCII. A representative scatter plot is shown (a) with analysis performed on cells negative for 7AAD-R1 (b) and double positive for CD11b and F4/80 (c). Levels of CD80, CD86 and MHC class II were then measured on these cells with a representative histogram showing CD80 expression on untreated cells (ﬁlled histogram) compared to its relevant isotype control (unﬁlled histogram) (d). Geomeans for unstimulated, E, J or S isolate antigen and LPS stimulated BMMΦ and relevant isotype controls were calculated and a fold change for Geomean anti cell surface marker antibody/Geomean Isotype control antibody was plotted for CD80, CD86 and MHCII expression on F480+CD11b+7AAD−cells (e). Experiments were performed four times and data shown are a representative experiment. those two occasions the S isolate E/S increased levels of CD80 and CD86 but decreased levels of MHC class II compared to the E isolate and J isolate E/S (Figure 1e), although unstim- ulated cultures had a geomean ratio for CD80 expression above values of cells cultured with isolate speciﬁc E/S and LPS. Signiﬁcantly higher levels of IL-10 and IL-6 are secreted by BMMΦ following stimulation with S isolate E/S compared to E or J isolate E/S Following the culture of BMMΦ for 48 h with medium alone, E isolate E/S, J isolate E/S, S isolate E/S or LPS the supernatants were analysed for a panel of cytokines. S isolate E/S signiﬁcantly increased levels of IL-10 and IL-6 cytokines in the culture medium compared to E/S from the two other isolates (Figure 2a,d). This signiﬁcant elevation was consistent in all four experiments with levels of both these cytokines undetectable in the unstimulated controls. In addition, levels of TGF-β were signiﬁcantly decreased following stimulation with S isolate E/S compared to levels of this cytokine produced by BMMΦ stimulated with E iso- late or J isolate E/S (Figure 2c). Levels of IL-12p70, TNF-α and MIP1-α did not differ signiﬁcantly between the isolates (Figure 2b,e,f, respectively). Further, levels of TNF-α and IL-12p70 secreted by BMMΦ following addition of isolate- 126 speciﬁc E/S were not signiﬁcantly different to unstimulated (medium alone) samples. In contrast, levels of MIP1-α were elevated from unstimulated levels to levels equivalent to the LPS control for all three isolates (Figure 2e). In addition to these cytokines presented, levels of Th2-associated cytokines IL-4, IL-5, IL-9 and IL-13 were analysed and found to be undetectable (data not shown). Exposure of BMDCs to isolate E/S results in elevated levels of CD40 expression BMDCs were cultured for 48 h with isolate-speciﬁc E/S or LPS and analysed for the expression of CD80, CD86, CD40 and MHC class II. 7AAD+ cells were classed as dead and removed from fur- ther analysis (Figure 3b). 7AAD negative cells were stained for DC phenotype markers CD11c and CD11b to assess purity of the culture prior to downstream analysis of co- stimulatory markers and MHC class II (Figure 3b,c). The Percentage of CD11c+CD11b+ cells ranged from 55% to 65% across all four experiments. In addition, separate samples were stained for F4/80 to exclude any macrophage contamination. In all cases cells were F4/80 negative (data not shown). Levels of CD80, CD86, CD40 and MHC class II were assessed individually on cells that were CD11c+CD11b+7AAD–. Levels of cell surface marker © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Parasite Immunology, 31, 123–131