Fluostar omega luminometer

To determine anti-OVA IgE and IgG1 levels, white Costar^® 96-well plates were coated with 10 µg/ml OVA in Tris/saline buffer (pH 7.2). Briefly, wells were washed and subsequently blocked with 2% skim milk solution. Standards (IgE (Serotec) and IgG1 (Sigma-Aldrich)) were serially diluted 1:2, starting at 200 ng/ml or 500 ng/ml, respectively. After incubation with standards and diluted samples, wells were repeatedly washed and subsequently incubated with biotinylated anti-mouse IgE (BD) or anti-mouse IgG1 (Southern Biotech). Wells were then washed and incubated with Streptavidin-HRP (Thermo Scientific). Following further washing, substrate (Thermo Scientific) was added to the wells and responses were immediately measured with a Luminometer (FlUOstar Omega, BMG Labtech). For quantification of serum MMCP-1, serum was obtained 4 h after oral gavage (o.g.) challenge. MMCP-1 concentrations were determined by ELISA following the manufacturer’s instructions (eBiosciences).

Unless otherwise stated all bioluminescence reagents are from Promega. Transiently transfected parasites were lysed with 0.1% saponin and washed twice in PBS to remove the hemoglobin. For firefly luciferase, parasite pellets were re-suspended in 20 µL of 1x Luciferase Cell Culture Lysis Reagent, mixed with 100 µL of Luciferase Assay Reagent, containing the Luciferase Assay Substrate Luciferin previously dissolved in Luciferase Assay Buffer, and immediately measured for 30 seconds in a FLUOstar Omega Luminometer (BMG Labtech) with the gain adjusted to maximum. For Nluc, parasite pellets were re-suspended in 100 µL of PBS, mixed with 100 µL of 2x Nano-Glo Luciferase Assay Reagent and measured in the luminometer with the same gain and for the same time used for firefly luciferase. Nano-Glo Luciferase Assay Reagent was made by adding one volume of Nano-Glo Luciferase Assay Substrate to 50 volumes of Nano-Glo Luciferase Assay Buffer as recommended by the manufacturer. Cultures of stably transfected Nluc parasites, were diluted in RPMI to 0.1% hematocrit, mixed with 1 volume of Nano-Glo Luciferase Assay Reagent and measured for 2–10 seconds as indicated in each experiment in a luminometer with the gain reduced by 10% for the sample with the highest signal on the plate to prevent saturation.