V plex custom mouse cytokine assay

Serum levels of IFN-γ, IL-10 and TNF-α were determined using the V-PLEX Custom Mouse Cytokine assay (Meso Scale Discovery, Rockville, USA) according to the manufacturer’s instructions.

Blood was collected from CO₂ euthanized mice via cardiac puncture, centrifuged (15 minutes, 10.000xg, 25°C), and serum was kept at -20°C. Serum levels of IFN-γ, IL-6, IL-10, IL-12p70, TNF and KC (CXCL1) were determined using the V-PLEX Custom Mouse Cytokine assay (Meso Scale Discovery, Maryland, USA). Serum MIF levels were measured by ELISA as recommended by the suppliers (R&D Systems). Alternatively, culture medium concentrations of MIF, TNF, CCL2 and KC (R&D Systems) as well as IFN-γ, IL-6 and IL-10 (Pharmingen) were determined by ELISA as recommended by the suppliers. Parasite-specific IgG responses were determined using soluble lysate freshly prepared from DEAE-purified T. congolense parasites recovered from WT mice at the peak of infection (around day 7). Lysate was coated overnight at 10 μg/ml PBS in 96-well Maxisorp plates (NUNC). Plates were washed (0.1% Tween 20 in PBS) and blocked (1% BSA in PBS) for 1 hour. Next, plates were washed and the sera (100 μl) serially diluted starting from 1/100 in blocking buffer were added. The ELISA was subsequently performed as described by the suppliers (SBA Clonotyping system-HRP kit (SouthernBiotech, USA)). As negative controls, blood samples incubated on lysate-free plates were used. The OD_450nm recorded on lysate-free plates was subtracted from the OD_450nm recorded on lysate-coated plates.

Tail vein blood samples were collected in heparinized capillary tubes and plasma was collected following centrifugation (15 minutes, 10.000 × g, 4 °C). Plasma levels of IFN-γ, IL-6, IL-10, IL-12p70, IL1β, TNF-α and CXCL1 were determined using the V-PLEX Custom Mouse Cytokine assay (Meso Scale Discovery, Maryland, USA).