Tumor necrosis factor (tnf)

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.

Cells were plated at 5 × 10⁴ cells per well in 96‐well plates coated with Matrigel. To control the number of cells, the assay was performed relatively soon after seeding. Therefore, the seeding density was higher than that in other assays or usual passages. Three days after plating, culture media were replaced with 100 μL/well of Astrocyte Media containing IL‐1β (Peprotech) or TNFα (Peprotech), harvested 24 h after replacement. Twenty‐five microlitres of conditioned media was subjected to electrochemiluminescence (ECL)‐ELISA analysis by using a V‐PLEX Custom Human Biomarkers kit (IL‐1β, IL‐6, TNF‐α, IL‐4 and GM‐CSF) (Meso Scale Discovery, Rockville, MD) and MESO SECTOR S600 (Meso Scale Discovery). Cytokine concentrations were calculated based on the kit's standard calibration curve using a four‐parameter logistic fit by applying MSD Discovery workbench 4.0 software (Meso Scale Discovery).