Volume 31, Number 3, March 2009 S isolate antigens stimulate APC derived IL-10 and IL-6 Protocol The BMMΦ at 1 × 106/mL or BMDC at 1 × 106/mL were added to 12-well plates and cultured for 48 h in media 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 same batch of E/S from each of the three isolates and LPS was used for all four independent BMMΦ and four independent BMDC experiments to minimize any batch to batch differences. Supernatants were assessed using a custom cytometric bead array kit (CBA- BD biosciences) and cells were collected for FACs analysis. Four wells of BMMΦ or BMDC were cultured with each condition. The cells from each of the four wells were combined for FACs analysis, but supernatants were kept separate and analysed individually. This was repeated a further three times. Cytokine bead array (CBA) Supernatants were assessed using a custom cytometric bead array kit (CBA-BD biosciences) for IL-10, IL-12p70, IFN-γ, TGF-β, IL-6, TNF-α, MCP-1 and MIP-1α. Brieﬂy samples were incubated with the combined capture bead cocktail, diluted in capture bead diluent and incubated for 1 h at RT on a digital shaker at 500 r.p.m. Following incubation, samples and beads were mixed with the combined cocktail of PE detection antibodies for the eight cytokines and incubated for 1 h. Sample was then washed and resuspended in FACS buffer. Cytokine concentrations were measured via quantiﬁcation of PE ﬂuorescence of samples in reference to a standard curve. Flow cytometric analysis BMMΦ and BMDCs were stained as a single suspension in 96 well plates. Cells were stained at a concentration of 1 × 106 cells per 200 μL FACs buffer (0·1% BSA, 0·05% Sodium Azide in PBS). Plates were washed in FACS buffer and spun at 3000 g for 5 min between each staining step and twice before acquisition. Non-speciﬁc antibody binding was prevented via incubation at 4°C with 0·25 μg anti-Fc-γ Receptor I/II (CD16/CD32) antibodies (BD Biosciences). Cells were then stained for either CD80-biotin (16-10A1), CD86- biotin (GL1), CD40-biotin (1C10) or MHC class II- biotin (M5/114·15·12) by incubation for 30 min at 4°C. A second antibody step consisted of adding a master mix of CD11b-PE (M1/70), F4/80-FITC (6F12) or CD11c-FITC (HL3), 7AAD and SA-APC for a further 30 min at 4°C in the dark. Relevant isotype controls were added for each antibody to duplicate samples. Cells were ﬁnally resuspended in equal quantities of FACS buffer and FACS Fix (2% formalydehyde in PBS). Samples were analysed on FACS- Calibur (Becton Dickinson) using CellQuest Pro software (BD Biosciences, Mountain View, CA). Geomean levels were recorded for samples stained for surface markers and isotype control samples to generate a fold-change ratio of geomean surface marker/geomean isotype control. Statistics Cytokine results are expressed as means ± standard deviation (SD). Statistical analysis was performed using the one-way avano with a Bonferroni post-test. Signiﬁcant differences on graphs are only shown between the isolates for clarity. All statistical tests were performed using GraphPad Prism software (San Diego, CA). RESULTS Isolate-speciﬁc E/S fail to consistently alter BMMΦ cell surface expression of CD80, CD86 and MHC class II BMMΦ were cultured with media alone, E isolate E/S, J isolate E/S, S isolate E/S or Lipopolysaccharide (LPS) as a positive control. Following 48 h of culture, cells were stained for cell surface markers associated with a macrophage phe- notype F4/80, CD11b and cell viability marker 7AAD in addition to the co-stimulatory markers CD80, CD86 and MHC class II. Dead cells were eliminated from the analysis by grouping on cells negative for 7AAD (Figure 1b). The remaining analysis was then done on cells that were double positive for F4/80 and CD11b (Figure 1c), with the double positive quadrant determined by the isotype control proﬁle (data not shown). The percentage of BMMΦ positive for both F4/80 and CD11b ranged from 60% to 70% for each of the four experiments. In addition separate BMMΦ samples were taken and analysed for the level of CD11c expression. Samples that were F4/80+ or CD11b+or F4/80+CD11b+ were all < 1% positive for CD11c (data not shown). Cells that were F4/80+CD11b+7AAD– were analysed for CD80, CD86 and MHC class II expression. The level of expression of CD80, CD86 and MHC class II was calculated using the geomean levels and comparing them to their relevant individual isotype controls. An exam- ple of CD80 expression on F4/80+CD11b+7AAD– cells of a positive sample compared to an isotype sample is shown (Figure 1d). The geomean of the sample was divided by the geomean of the isotype controls and is therefore shown as a fold change in Figure 1(e). No consistent changes in the geomean ratios for CD80, CD86 or MHC class II were seen on BMMΦ that were F4/ 80+CD11b+7AAD– (Figure 1e). Examples shown are values that were consistent on two out of the four experiments. On © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Parasite Immunology, 31, 123–131 125